U.S. patent number 10,563,234 [Application Number 15/361,645] was granted by the patent office on 2020-02-18 for method for producing l-amino acids.
This patent grant is currently assigned to AJINOMOTO CO., INC.. The grantee listed for this patent is AJINOMOTO CO., INC.. Invention is credited to Hidetaka Doi, Akiko Matsudaira, Yoshihiro Usuda.
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United States Patent |
10,563,234 |
Doi , et al. |
February 18, 2020 |
Method for producing L-amino acids
Abstract
A method for producing an L-amino acid is provided. An L-amino
acid is produced by culturing a bacterium belonging to the family
Enterobacteriaceae and having an L-amino acid-producing ability,
wherein the bacterium has been modified so that the activity of
aconitase is increased, or the activities of aconitase and
acetaldehyde dehydrogenase are increased, in a medium, and
collecting the L-amino acid from the medium or cells of the
bacterium.
Inventors: |
Doi; Hidetaka (Kanagawa,
JP), Matsudaira; Akiko (Kanagawa, JP),
Usuda; Yoshihiro (Kanagawa, JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
AJINOMOTO CO., INC. |
Tokyo |
N/A |
JP |
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Assignee: |
AJINOMOTO CO., INC. (Tokyo,
JP)
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Family
ID: |
54766818 |
Appl.
No.: |
15/361,645 |
Filed: |
November 28, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170073714 A1 |
Mar 16, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/JP2015/066072 |
Jun 3, 2015 |
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Foreign Application Priority Data
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Jun 3, 2014 [JP] |
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2014-114799 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/09 (20130101); C12N 15/00 (20130101); C12P
13/08 (20130101) |
Current International
Class: |
C12P
13/04 (20060101); C12P 13/08 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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103173504 |
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Jun 2013 |
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CN |
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2192170 |
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Jun 2010 |
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EP |
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2290092 |
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Mar 2011 |
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EP |
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2014-506466 |
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Mar 2014 |
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JP |
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WO-03076629 |
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Sep 2003 |
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WO |
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WO2008/010565 |
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Jan 2008 |
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WO |
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WO2009/031565 |
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Mar 2009 |
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WO |
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WO2010/101053 |
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Sep 2010 |
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WO |
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WO2011/096554 |
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Aug 2011 |
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WO |
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WO2012/002486 |
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Jan 2012 |
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WO |
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WO2012/077739 |
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Jun 2012 |
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WO |
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Other References
Zhou et al., Cell Mol Life Sci 63:2260-2290, 2006 (Year: 2006).
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Kozak, M., Gene 234:187-208, 1999 (Year: 1999). cited by examiner
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2014). cited by examiner .
UniProt Database Accession No. P36683, May 2014, 5 pages (Year:
2014). cited by examiner .
Uni Prot Database Accession No. P37685, May 2014, 3 pages (Year:
2014). cited by examiner .
Schendel, P., "Current Protocols in Molecular Biology" (1998)
16.1.1-16.1.3 (Year: 1998). cited by examiner .
Tang et al., Microbiology 148:1027-1037, 2002 (Year: 2002). cited
by examiner .
International Search Report for PCT Patent App. No.
PCT/JP2015/066072 (dated Aug. 4, 2015). cited by applicant .
Ho, K. K., et al., "Isolation and Characterization of an Aldehyde
Dehydrogenase Encoded by the aldB Gene of Escherichia coli," J.
Bacteriol. 2005;187:1067-1073. cited by applicant .
Cunningham, L., et al., "Transcriptional regulation of the
aconitase genes (acnA and acnB) of Escherichia coli," Microbiol.
1997;143:3795-3805. cited by applicant .
International Preliminary Report on Patentability for PCT Patent
App. No. PCT/JP2015/066072 (dated Dec. 15, 2016). cited by
applicant .
Supplementary European Search Report for European Patent App. No.
15803550.1 (dated Oct. 19, 2017). cited by applicant.
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Primary Examiner: Steadman; David
Attorney, Agent or Firm: Cermak Nakajima & McGowan LLP
Cermak; Shelly Guest
Parent Case Text
This application is a Continuation of, and claims priority under 35
U.S.C. .sctn. 120 to, International Application No.
PCT/JP2015/066072, filed Jun. 3, 2015, and claims priority
therethrough under 35 U.S.C. .sctn. 119 to Japanese Patent
Application No. 2014-114799, filed Jun. 3, 2014, the entireties of
which are incorporated by reference herein. Also, the Sequence
Listing filed electronically herewith is hereby incorporated by
reference (File name: 2016-11-28T_US-558_Seq_List; File size: 158
KB; Date recorded: Nov. 28, 2016).
Claims
The invention claimed is:
1. A method for producing an L-amino acid, the method comprising:
A) culturing an Enterobacteriaceae bacterium in a medium comprising
ethanol as a carbon source, resulting in production and
accumulation of the L-amino acid in the medium or cells of the
bacterium, and B) collecting the L-amino acid from the medium or
the cells of the bacterium; wherein the bacterium has been modified
to increase the activities of aconitase and acetaldehyde
dehydrogenase B (AldB) as compared with a corresponding
non-modified Enterobacteriaceae bacterium, wherein the activity of
the aconitase is increased by increasing the copy number of a gene
encoding the aconitase, by modifying an expression control sequence
of the gene encoding the aconitase, or by a combination thereof,
wherein the activity of the AldB is increased by increasing the
copy number of a gene encoding the AldB, by modifying an expression
control sequence of the gene encoding the AldB, or by a combination
thereof, and wherein the bacterium has an L-amino acid-producing
ability in a medium comprising ethanol and is able to aerobically
utilize ethanol as a carbon source.
2. The method according to claim 1, wherein the aconitase is an
aconitase A (AcnA) protein or aconitase B (AcnB) protein.
3. The method according to claim 2, wherein the AcnA protein is a
protein selected from the group consisting of: (a) a protein
comprising the amino acid sequence of SEQ ID NO: 22, 24, 26, or 28,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 22,
24, 26, or 28, but wherein said sequence includes substitution,
deletion, insertion, or addition of at least 1 and no more than 10
amino acid residues, and wherein said protein has aconitase
activity, and (c) a protein comprising an amino acid sequence
having an identity of 90% or higher to the amino acid sequence of
SEQ ID NO: 22, 24, 26, or 28, and wherein said protein has
aconitase activity.
4. The method according to claim 2, wherein the AcnB protein is a
protein selected from the group consisting of: (a) a protein
comprising the amino acid sequence of SEQ ID NO: 30, 32, 34, or 36,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 30,
32, 34, or 36, but wherein said sequence includes substitution,
deletion, insertion, or addition of at least 1 and no more than 10
amino acid residues, and wherein said protein has aconitase
activity, and (c) a protein comprising an amino acid sequence
having an identity of 90% or higher to the amino acid sequence of
SEQ ID NO: 30, 32, 34, or 36, and wherein said protein has
aconitase activity.
5. The method according to claim 1, wherein the AldB protein is a
protein selected from the group consisting of: (a) a protein
comprising the amino acid sequence of SEQ ID NO: 38, 40, 42, or 44,
(b) a protein comprising the amino acid sequence of SEQ ID NO: 38,
40, 42, or 44, but wherein said sequence includes substitution,
deletion, insertion, or addition of at least 1 and no more than 10
amino acid residues, and wherein said protein has acetaldehyde
dehydrogenase B activity, and (c) a protein comprising an amino
acid sequence having an identity of 90% or higher to the amino acid
sequence of SEQ ID NO: 38, 40, 42, or 44, and wherein said protein
has acetaldehyde dehydrogenase B activity.
6. The method according to claim 1, wherein the bacterium has been
further modified to increase an activity of an ethanol metabolic
enzyme as compared with a corresponding non-modified
Enterobacteriaceae bacterium by increasing the copy number of a
gene encoding the ethanol metabolic enzyme, by modifying an
expression control sequence of a gene encoding the ethanol
metabolic enzyme, or by a combination thereof, and wherein the
ethanol metabolic enzyme is selected from the group consisting of
alcohol dehydrogenase, CoA-dependent acetaldehyde dehydrogenase,
and combinations thereof.
7. The method according to claim 1, wherein the bacterium has been
transformed with a polynucleotide encoding a mutant alcohol
dehydrogenase E (AdhE) protein, and wherein the mutant AdhE protein
has alcohol dehydrogenase activity and CoA-dependent acetaldehyde
dehydrogenase activity and comprises the amino acid sequence of SEQ
ID NO: 46, except for replacement of an amino acid residue
corresponding to the glutamic acid residue at position 568 in the
amino acid sequence of SEQ ID NO: 46 with an amino acid residue
other than glutamic acid and aspartic acid, and optionally an
additional mutation selected from the group consisting of: (A)
replacement of an amino acid residue corresponding to the glutamic
acid residue at position 560 in the amino acid sequence of SEQ ID
NO: 46 with another amino acid residue, (B) replacement of an amino
acid residue corresponding to the phenylalanine residue at position
566 in the amino acid sequence of SEQ ID NO: 46 with another amino
acid residue, (C) replacement of amino acid residues corresponding
to the glutamic acid residue at position 22, methionine residue at
position 236, tyrosine residue at position 461, isoleucine residue
at position 554, and alanine residue at position 786 in the amino
acid sequence of SEQ ID NO: 46 with other amino acid residues, and
(D) combinations thereof.
8. The method according to claim 7, wherein the replacement of an
amino acid residue corresponding to the glutamic acid residue at
position 568 in the amino acid sequence of SEQ ID NO: 46 is with
lysine.
9. The method according to claim 1, wherein the bacterium is an
Escherichia bacterium.
10. The method according to claim 9, wherein the bacterium is
Escherichia coli.
11. The method according to claim 1, wherein the L-amino acid is
L-lysine.
12. The method according to claim 11, wherein the bacterium further
has a characteristic selected from the group consisting of: (A) the
bacterium has been modified to increase activity or activities of
an enzyme selected from the group consisting of dihydrodipicolinate
reductase, diaminopimelate decarboxylase, diaminopimelate
dehydrogenase, phosphoenolpyruvate carboxylase, aspartate
aminotransferase, diaminopimelate epimerase, aspartate semialdehyde
dehydrogenase, tetrahydrodipicolinate succinylase,
succinyldiaminopimelate deacylase, and combinations thereof as
compared with a corresponding non-modified Enterobacteriaceae
bacterium by increasing the copy number of a gene encoding the
selected enzyme, by modifying an expression control sequence of the
selected enzyme, or by a combination thereof, (B) the bacterium has
been modified to reduce activity of lysine decarboxylase as
compared with a corresponding non-modified Enterobacteriaceae
bacterium by disrupting a gene encoding the lysine decarboxylase,
and (C) combinations thereof.
Description
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a method for producing an L-amino
acid utilizing a bacterium. L-amino acids are industrially useful
as additives for animal feeds, ingredients of seasonings,
ingredients of foods and drinks, amino acid infusions, and so
forth.
Brief Description of the Related Art
L-Amino acids are industrially produced by, for example,
fermentation using various microorganisms having an L-amino
acid-producing ability. Examples of such methods for producing an
L-amino acid by fermentation include, for example, methods of using
a wild-type microorganism (wild-type strain), methods of using an
auxotrophic strain derived from a wild-type strain, methods of
using a metabolic regulation mutant strain derived from a wild-type
strain as a mutant strain resistant to any of various drugs, and
methods of using a strain having properties as both auxotrophic
strain and metabolic regulation mutant strain.
In recent years, microorganisms in which an L-amino acid-producing
ability is improved by recombinant DNA techniques are also utilized
for production of L-amino acids. Examples of method for improving
an L-amino acid-producing ability of a microorganism include, for
example, enhancing the expression of a gene encoding an L-amino
acid biosynthesis system enzyme (U.S. Pat. Nos. 5,168,056 and
5,776,736), and enhancing inflow of a carbon source into an L-amino
acid biosynthesis system (U.S. Pat. No. 5,906,925).
In the conventional industrial production of objective substances
such as L-amino acids by fermentation, glucose, fructose, sucrose,
blackstrap molasses, starch hydrolysate, and so forth have been
used as a carbon source.
It is also possible to use alcohols such as ethanol as a carbon
source. As methods for producing an L-amino acid by fermentation
using ethanol as a carbon source, there are known, for example, a
method of using an Enterobacteriaceae bacterium modified so that it
expresses alcohol dehydrogenase under aerobic conditions
(WO2008/010565), a method of using an Enterobacteriaceae bacterium
modified so that the activity of pyruvate synthase or
pyruvate:NADP.sup.+ oxidoreductase is increased (WO2009/031565), a
method of using an Enterobacteriaceae bacterium modified so that
the activity of ribonuclease G is reduced (WO2010/101053), a method
of using an Enterobacteriaceae bacterium modified so that it
harbors a mutant ribosome S1 protein (WO2011/096554), a method of
using an Enterobacteriaceae bacterium modified so that the activity
of an A1 dB protein is reduced (WO2012/002486), and a method of
using an Enterobacteriaceae bacterium modified so that the
intracellular concentration of hydrogen peroxide is reduced
(Japanese Patent Laid-open (Kokai) No. 2014-036576).
Aconitase is a dehydratase/hydratase that reversibly catalyzes the
isomerization between citrate and isocitrate in the TCA cycle or
glyoxylate cycle (EC 4.2.1.3). Escherichia coli has at least two
kinds of isozymes of aconitase, AcnA and AcnB. The identity of the
amino acid sequences of AcnA and AcnB is about 17%. AcnB is the
major aconitase of Escherichia coli, and is expressed especially in
the logarithmic phase (Cunningham L1, Gruer M J, Guest J R.,
Microbiology., 1997, December; 143(12):3795-805). On the other
hand, AcnA is induced by iron or oxidization stress, and is
expressed especially in the resting stage (Ho K K, Weiner H., J.
Bacteriol., 2005, February; 187(3): 1067-73).
Acetaldehyde dehydrogenase is an enzyme that reversibly catalyzes
the reaction of generating acetic acid from acetaldehyde by using
NAD.sup.+ or NADP.sup.+ as an electron acceptor (EC 1.2.1.3, EC
1.2.1.4, EC 1.2.1.5, EC 1.2.1.22, etc.). For example, the AldB
protein of Escherichia coli has the acetaldehyde dehydrogenase
activity that uses NADP.sup.+ as an electron acceptor. As described
above, it is known that a reduction of the activity of the AldB
protein is effective for L-amino acid production using ethanol as a
carbon source.
SUMMARY OF THE INVENTION
Aspects to be Achieved by the Invention
An aspect of the present invention is to develop a novel technique
for improving an L-amino acid-producing ability of a bacterium, and
thereby provide a method for efficiently producing an L-amino
acid.
It has been found that by modifying a bacterium so that the
activity of aconitase is increased, or both the activities of
aconitase and acetaldehyde dehydrogenase are increased, L-amino
acid production by the bacterium using ethanol as a carbon source
can be improved.
It is an aspect of the present invention to provide a method for
producing an L-amino acid, the method comprising (A) culturing an
Enterobacteriaceae bacterium and having an L-amino acid-producing
ability in a medium comprising ethanol, resulting in the production
and accumulation of the L-amino acid in the medium or cells of the
bacterium; and (B) collecting the L-amino acid from the medium or
the cells, wherein the bacterium has been modified to increase the
activity of aconitase, and wherein the aconitase is an AcnB
protein.
It is a further aspect of the present invention to provide the
method as described above, wherein the AcnB protein is selected
from the group consisting of (a) a protein comprising the amino
acid sequence of SEQ ID NO: 30, 32, 34, or 36; (b) a protein
comprising the amino acid sequence of SEQ ID NO: 30, 32, 34, or 36,
but wherein said sequence includes substitution, deletion,
insertion, or addition of 1 to 10 amino acid residues, and said
protein has aconitase activity; (c) a protein comprising an amino
acid sequence having an identity of 90% or higher to the amino acid
sequence of SEQ ID NO: 30, 32, 34, or 36, and wherein said protein
has aconitase activity.
It is a further aspect of the present invention to provide a method
for producing an L-amino acid, the method comprising (A) culturing
an Enterobacteriaceae bacterium having an L-amino acid-producing
ability in a medium comprising ethanol, resulting in production and
accumulation of the L-amino acid in the medium or cells of the
bacterium; and (B) collecting the L-amino acid from the medium or
the cells, wherein the bacterium has been modified to increase the
activities of aconitase and acetaldehyde dehydrogenase.
It is a further aspect of the present invention to provide the
method as described above, wherein the aconitase is an AcnA protein
or AcnB protein.
It is a further aspect of the present invention to provide the
method as described above, wherein the AcnA protein is selected
from the group consisting of (a) a protein comprising the amino
acid sequence of SEQ ID NO: 22, 24, 26, or 28; (b) a protein
comprising the amino acid sequence of SEQ ID NO: 22, 24, 26, or 28,
but wherein said sequence includes substitution, deletion,
insertion, or addition of 1 to 10 amino acid residues, and wherein
said protein has aconitase activity; (c) a protein comprising an
amino acid sequence having an identity of 90% or higher to the
amino acid sequence of SEQ ID NO: 22, 24, 26, or 28, and wherein
said protein has aconitase activity.
It is a further aspect of the present invention to provide the
method as described above, wherein the AcnB protein is selected
from the group consisting of (a) a protein comprising the amino
acid sequence of SEQ ID NO: 30, 32, 34, or 36; (b) a protein
comprising the amino acid sequence of SEQ ID NO: 30, 32, 34, or 36,
but wherein said sequence includes substitution, deletion,
insertion, or addition of 1 to 10 amino acid residues, and wherein
said protein has aconitase activity; (c) a protein comprising an
amino acid sequence having an identity of 90% or higher to the
amino acid sequence of SEQ ID NO: 30, 32, 34, or 36, and wherein
said protein has aconitase activity.
It is a further aspect of the present invention to provide the
method as described above, wherein the acetaldehyde dehydrogenase
is an AldB protein.
It is a further aspect of the present invention to provide the
method as described above, wherein the AldB protein is selected
from the group consisting of (a) a protein comprising the amino
acid sequence of SEQ ID NO: 38, 40, 42, or 44; (b) a protein
comprising the amino acid sequence of SEQ ID NO: 38, 40, 42, or 44,
but wherein said sequence includes substitution, deletion,
insertion, or addition of 1 to 10 amino acid residues, and wherein
said protein has acetaldehyde dehydrogenase activity; (c) a protein
comprising an amino acid sequence having an identity of 90% or
higher to the amino acid sequence of SEQ ID NO: 38, 40, 42, or 44,
and wherein said protein has acetaldehyde dehydrogenase
activity.
It is a further aspect of the present invention to provide the
method as described above, wherein the bacterium has been further
modified to increase the activity of an ethanol metabolic
enzyme.
It is a further aspect of the present invention to provide the
method as described above, wherein the bacterium is able to
aerobically utilize ethanol.
It is a further aspect of the present invention to provide the
method as described above, wherein the bacterium has been modified
to harbor a mutant adhE gene, and wherein the mutant adhE gene is
encodes a mutant AdhE protein comprising a mutation that results in
improved resistance to inactivation under aerobic conditions.
It is a further aspect of the present invention to provide the
method as described above, wherein the mutation is replacement of
an amino acid residue corresponding to the glutamic acid residue at
position 568 in the amino acid sequence of SEQ ID NO: 46 with an
amino acid residue other than glutamic acid and aspartic acid.
It is a further aspect of the present invention to provide the
method as described above, wherein the amino acid residue other
than glutamic acid and aspartic acid is lysine.
It is a further aspect of the present invention to provide the
method as described above, wherein the mutant AdhE protein further
has an additional mutation selected from the group consisting of
(A) replacement of an amino acid residue corresponding to the
glutamic acid residue at position 560 in the amino acid sequence of
SEQ ID NO: 46 with another amino acid residue, (B) replacement of
an amino acid residue corresponding to the phenylalanine residue at
position 566 in the amino acid sequence of SEQ ID NO: 46 with
another amino acid residue, (C) replacement of amino acid residues
corresponding to the glutamic acid residue at position 22,
methionine residue at position 236, tyrosine residue at position
461, isoleucine residue at position 554, and alanine residue at
position 786 in the amino acid sequence of SEQ ID NO: 46 with other
amino acid residues; and (D) combinations thereof.
It is a further aspect of the present invention to provide the
method as described above, wherein the bacterium is an Escherichia
bacterium.
It is a further aspect of the present invention to provide the
method as described above, wherein the bacterium is Escherichia
coli.
It is a further aspect of the present invention to provide the
method as described above, wherein the L-amino acid is
L-lysine.
It is a further aspect of the present invention to provide the
method as described above, wherein the bacterium further has a
characteristic selected from the group consisting of (A) the
bacterium has been modified to increase the activity or activities
of an enzymes selected from the group consisting of
dihydrodipicolinate reductase, diaminopimelate decarboxylase,
diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase,
aspartate aminotransferase, diaminopimelate epimerase, aspartate
semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase,
succinyldiaminopimelate deacylase, and combinations thereof (B) the
bacterium has been modified to reduce the activity of lysine
decarboxylase; and (C) combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B: Alignment of amino acid sequences of various AldB
proteins
FIGS. 2A, 2B, and 2C: Alignment of amino acid sequences of various
AldE proteins.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereafter, the present invention will be explained in detail.
The method of the present invention is a method for producing an
L-amino acid by culturing an Enterobacteriaceae bacterium having an
L-amino acid-producing ability in a medium containing ethanol
resulting in production and accumulation of the L-amino acid in the
medium or cells of the bacterium, and collecting the L-amino acid
from the medium or the cells, wherein the bacterium has been
modified so that the activity of aconitase is increased, or the
activities of aconitase and acetaldehyde dehydrogenase are
increased. The bacterium used for this method is also referred to
as "the bacterium of the present invention".
<1> Bacterium of the Present Invention
The bacterium can belong to the family Enterobacteriaceae and also
can have an L-amino acid-producing ability. The bacterium has been
modified so that the activity of aconitase is increased, or the
activities of aconitase and acetaldehyde dehydrogenase are
increased.
<1-1> Bacterium Having L-Amino Acid-Producing Ability
The phrase "bacterium having an L-amino acid-producing ability"
refers to a bacterium having an ability to generate or produce, and
accumulate an objective L-amino acid in a medium or cells of the
bacterium to such a degree that the L-amino acid can be collected,
when the bacterium is cultured in the medium. The bacterium having
an L-amino acid-producing ability may be able to accumulate an
objective L-amino acid in a medium in an amount larger than that
obtainable with a non-modified strain. Examples of the non-modified
strain include a wild-type strain and parental strain. The
bacterium having an L-amino acid-producing ability may be a
bacterium that can accumulate an objective L-amino acid in a medium
in an amount of 0.5 g/L or more, or 1.0 g/L or more.
Examples of the L-amino acid include basic amino acids such as
L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline;
aliphatic amino acids such as L-isoleucine, L-alanine, L-valine,
L-leucine, and glycine; amino acids which are
hydroxy-monoaminocarboxylic acids such as L-threonine and L-serine;
cyclic amino acids such as L-proline; aromatic amino acids such as
L-phenylalanine, L-tyrosine, and L-tryptophan; sulfur-containing
amino acids such as L-cysteine, L-cystine, and L-methionine; acidic
amino acids such as L-glutamic acid and L-aspartic acid; and amino
acids having an amide group in the side chain such as L-glutamine
and L-asparagine. The bacterium can have an ability to produce a
single kind of L-amino acid, or two or more kinds of L-amino
acids.
Amino acids may be L-amino acids unless otherwise stated.
Furthermore, the L-amino acid to be produced may be in the form of
a free compound, a salt, or a mixture of these forms. That is, the
term "L-amino acid" can refer to an L-amino acid in a free form,
its salt, or a mixture of these, unless otherwise stated. Examples
of the salt will be described later.
Examples of bacteria belonging to the family Enterobacteriaceae
include bacteria belonging to the genus Escherichia, Enterobacter,
Pantoea, Klebsiella, Serratia, Envinia, Photorhabdus, Providencia,
Salmonella, Morganella, or the like. Specifically, bacteria
classified into the family Enterobacteriaceae according to the
taxonomy used in the NCBI (National Center for Biotechnology
Information) database (ncbi.nlm.nih.gov) can be used.
The Escherichia bacterial species are not particularly limited, and
examples include species classified into the genus Escherichia
according to the taxonomy known to those skilled in the field of
microbiology. Examples of the Escherichia bacterium include, for
example, those described in the work of Neidhardt et al. (Backmann
B. J., 1996, Derivations and Genotypes of some mutant derivatives
of Escherichia coli K-12, pp. 2460-2488, Table 1, In F. D.
Neidhardt (ed.), Escherichia coli and Salmonella Cellular and
Molecular Biology, Second Edition, American Society for
Microbiology Press, Washington, D.C.). Examples of the Escherichia
bacterial species include, for example, Escherichia coli. Specific
examples of Escherichia coli strains include, for example,
Escherichia coli W3110 (ATCC 27325) and Escherichia coli MG1655
(ATCC 47076) derived from the prototype wild-type strain, K-12.
The Enterobacter bacteria are not particularly limited, and
examples include species classified into the genus Enterobacter
according to classification known to a person skilled in the art of
microbiology. Examples of the Enterobacter bacterium include, for
example, Enterobacter agglomerans and Enterobacter aerogenes.
Specific examples of Enterobacter agglomerans strains include, for
example, the Enterobacter agglomerans ATCC 12287. Specific examples
of Enterobacter aerogenes strains include, for example, the
Enterobacter aerogenes ATCC 13048, NBRC 12010 (Biotechnol Bioeng.,
2007, Mar. 27; 98(2):340-348), and AJ110637 (FERM BP-10955).
Examples the Enterobacter bacterial strains also include, for
example, the strains described in European Patent Application
Laid-open (EP-A) No. 0952221. In addition, Enterobacter agglomerans
also include some strains classified as Pantoea agglomerans.
The Pantoea bacteria are not particularly limited, and examples
include species classified into the genus Pantoea according to
classification known to a person skilled in the art of
microbiology. Examples of the Pantoea bacterial species include,
for example, Pantoea ananatis, Pantoea stewartii, Pantoea
agglomerans, and Pantoea citrea. Specific examples of Pantoea
ananatis strains include, for example, the Pantoea ananatis
LMG20103, AJ13355 (FERM BP-6614), AJ13356 (FERM BP-6615), AJ13601
(FERM BP-7207), SC17 (FERM BP-11091), and SC17(0) (VKPM B-9246).
Some strains of Enterobacter agglomerans were recently reclassified
into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, or
the like on the basis of nucleotide sequence analysis of 16S rRNA
etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)). The Pantoea
bacteria include those reclassified into the genus Pantoea as
described above.
Examples of the Envinia bacteria include Envinia amylovora and
Envinia carotovora. Examples of the Klebsiella bacteria include
Klebsiella planticola.
These strains are available from, for example, the American Type
Culture Collection (Address: P.O. Box 1549, Manassas, Va. 20108,
United States of America). That is, registration numbers are given
to the respective strains, and the strains can be ordered by using
these registration numbers (refer to atcc.org/). The registration
numbers of the strains are listed in the catalogue of the American
Type Culture Collection.
The bacterium may be a bacterium inherently having an L-amino
acid-producing ability, or may be a bacterium modified so that it
has an L-amino acid-producing ability. The bacterium having an
L-amino acid-producing ability can be obtained by imparting an
L-amino acid-producing ability to such a bacterium as mentioned
above, or by enhancing an L-amino acid-producing ability of such a
bacterium as mentioned above.
To impart or enhance an L-amino acid-producing ability, methods
conventionally employed in the breeding of amino acid-producing
strains of coryneform bacteria, Escherichia bacteria, and so forth
(see "Amino Acid Fermentation", Gakkai Shuppan Center (Ltd.), 1st
Edition, published May 30, 1986, pp. 77-100) can be used. Examples
of such methods include, for example, acquiring an auxotrophic
mutant strain, acquiring an L-amino acid analogue-resistant strain,
acquiring a metabolic regulation mutant strain, and constructing a
recombinant strain in which the activity of an L-amino acid
biosynthetic enzyme is enhanced. In the breeding of L-amino
acid-producing bacteria, one of the above-described properties such
as auxotrophy, analogue resistance, and metabolic regulation
mutation may be imparted alone, or two or three or more of such
properties may be imparted in combination. Also, in the breeding of
L-amino acid-producing bacteria, the activity of one of L-amino
acid biosynthetic enzymes may be enhanced alone, or the activities
of two or three or more of such enzymes may be enhanced in
combination. Furthermore, imparting property(s) such as auxotrophy,
analogue resistance, and metabolic regulation mutation can be
combined with enhancing the activity(s) of biosynthetic
enzyme(s).
An auxotrophic mutant strain, analogue-resistant strain, or
metabolic regulation mutant strain having an L-amino acid-producing
ability can be obtained by subjecting a parental strain or
wild-type strain to a typical mutagenesis treatment, and then
selecting a strain exhibiting autotrophy, analogue resistance, or a
metabolic regulation mutation, and having an L-amino acid-producing
ability from the obtained mutant strains. Examples of typical
mutagenesis treatments include irradiation of X-ray or ultraviolet
and a treatment with a mutation agent such as
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate
(EMS), and methyl methanesulfonate (MMS).
An L-amino acid-producing ability can also be imparted or enhanced
by enhancing the activity of an enzyme involved in biosynthesis of
an objective L-amino acid. An enzyme activity can be enhanced by,
for example, modifying a bacterium so that the expression of a gene
encoding the enzyme is enhanced. Methods for enhancing gene
expression are described in WO00/18935, EP 1010755 A, and so forth.
The detailed procedures for enhancing enzyme activity will be
described later.
Furthermore, an L-amino acid-producing ability can also be imparted
or enhanced by reducing the activity of an enzyme that catalyzes a
reaction branching away from the biosynthetic pathway of an
objective L-amino acid to generate a compound other than the
objective L-amino acid. The "enzyme that catalyzes a reaction
branching away from the biosynthetic pathway of an objective
L-amino acid to generate a compound other than the objective
L-amino acid" includes an enzyme involved in decomposition of the
objective amino acid. The method for reducing an enzyme activity
will be described later.
Hereafter, L-amino acid-producing bacteria and methods for
imparting or enhancing an L-amino acid-producing ability will be
specifically exemplified. All of the properties of the L-amino
acid-producing bacteria and modifications for imparting or
enhancing an L-amino acid-producing ability may be used
independently or in any appropriate combination.
<L-Glutamic Acid-Producing Bacteria>
Examples of methods for imparting or enhancing L-glutamic
acid-producing ability include, for example, a method of modifying
a bacterium so that the bacterium has an increased activity or
activities of one or more of the L-glutamic acid biosynthesis
enzymes. Examples of such enzymes include, but are not particularly
limited to, glutamate dehydrogenase (gdhA), glutamine synthetase
(glnA), glutamate synthase (gltBD), isocitrate dehydrogenase
(icdA), aconitate hydratase (acnA, acnB), citrate synthase (OA),
methylcitrate synthase (prpC), pyruvate carboxylase (pyc), pyruvate
dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF),
phosphoenolpyruvate synthase (ppsA), enolase (eno),
phosphoglyceromutase (pgmA, pgml), phosphoglycerate kinase (pgk),
glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate
isomerase (tpiA), fructose bisphosphate aldolase (fbp), glucose
phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd),
2-keto-3-deoxy-6-phosphogluconate aldolase (eda), and
transhydrogenase. Shown in the parentheses after the names of the
enzymes are genes encoding the enzymes (the same shall apply to the
same occasions hereafter). It is preferable to enhance the activity
or activities of one or more of, for example, glutamate
dehydrogenase, citrate synthase, phosphoenol pyruvate carboxylase,
and methylcitrate synthase.
Examples of strains belonging to the family Enterobacteriaceae and
modified so that the expression of the citrate synthase gene,
phosphoenolpyruvate carboxylase gene, and/or glutamate
dehydrogenase gene are increased include those disclosed in EP
1078989 A, EP 955368 A, and EP 952221 A. Furthermore, examples of
strains belonging to the family Enterobacteriaceae and modified so
that the expression of a gene of the Entner-Doudoroff pathway (edd,
eda) is increased include those disclosed in EP 1352966 B.
Examples of methods for imparting or enhancing L-glutamic
acid-producing ability also include, for example, a method of
modifying a bacterium so that the bacterium has a reduced activity
or activities of one or more enzymes that catalyze a reaction
branching away from the biosynthesis pathway of L-glutamine to
generate a compound other than L-glutamic acid. Examples of such
enzymes include, but are not particularly limited to, isocitrate
lyase (aceA), .alpha.-ketoglutarate dehydrogenase (sucA),
acetolactate synthase (ilvl), formate acetyltransferase (pfl),
lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), glutamate
decarboxylase (gadAB), and succinate dehydrogenase (sdhABCD). It is
preferable to reduce or delete, for example, the
.alpha.-ketoglutarate dehydrogenase activity.
Escherichia bacteria having a reduced .alpha.-ketoglutarate
dehydrogenase activity or deficient in the .alpha.-ketoglutarate
dehydrogenase activity, and methods for obtaining them are
described in U.S. Pat. Nos. 5,378,616 and 5,573,945. Furthermore,
methods for reducing or deleting the .alpha.-ketoglutarate
dehydrogenase activity of Enterobacteriaceae bacteria such as
Pantoea bacteria, Enterobacter bacteria, Klebsiella bacteria, and
Erwinia bacteria are disclosed in U.S. Pat. Nos. 6,197,559,
6,682,912, 6,331,419, 8,129,151, and WO2008/075483. Specific
examples of Escherichia bacteria having a reduced
.alpha.-ketoglutarate dehydrogenase activity or deficient in the
.alpha.-ketoglutarate dehydrogenase activity include the following
strains.
E. coli W3110sucA::Km.sup.r
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)
E. coli W3110sucA::Km.sup.r is a strain obtained by disrupting the
sucA gene encoding .alpha.-ketoglutarate dehydrogenase of E. coli
W3110. This strain is completely deficient in the
.alpha.-ketoglutarate dehydrogenase activity.
Examples of L-glutamic acid-producing bacteria and parental strains
that can be used to derive such bacteria also include Pantoea
bacteria, such as Pantoea ananatis AJ13355 (FERM BP-6614), Pantoea
ananatis SC17 (FERM BP-11091), and Pantoea ananatis SC17(0) (VKPM
B-9246). The AJ13355 strain is isolated from soil in Iwata-shi,
Shizuoka-ken, Japan as a strain that can proliferate in a low pH
medium containing L-glutamic acid and a carbon source. The SC17
strain is selected as a low phlegm-producing mutant strain from the
AJ13355 strain (U.S. Pat. No. 6,596,517). The SC17 strain was
deposited at the independent administrative agency, National
Institute of Advanced Industrial Science and Technology,
International Patent Organism Depository (currently independent
administrative agency, National Institute of Technology and
Evaluation, International Patent Organism Depositary, #120, 2-5-8
Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on Feb.
4, 2009, and assigned an accession number of FERM BP-11091. The
AJ13355 strain was deposited at the National Institute of
Bioscience and Human Technology, Agency of Industrial Science and
Technology (currently, independent administrative agency, National
Institute of Technology and Evaluation, International Patent
Organism Depositary, #120, 2-5-8 Kazusakamatari, Kisarazu-shi,
Chiba-ken, 292-0818, Japan) on Feb. 19, 1998 and assigned an
accession number of FERM P-16644. Then, the deposit was converted
to an international deposit under the provisions of Budapest Treaty
on Jan. 11, 1999, and assigned an accession number of FERM
BP-6614.
Furthermore, examples of L-glutamic acid-producing bacteria and
parental strains that can be used to derive such bacteria also
include Pantoea bacteria having a reduced .alpha.-ketoglutarate
dehydrogenase activity or deficient in the .alpha.-ketoglutarate
dehydrogenase activity. Examples of such strains include AJ13356
(U.S. Pat. No. 6,331,419), which is an .alpha.-ketoglutarate
dehydrogenase E1 subunit (sucA) gene-deficient strain of the
AJ13355 strain, and the SC17sucA strain (U.S. Pat. No. 6,596,517),
which is a sucA gene-deficient strain of the SC17 strain. The
AJ13356 strain was deposited at the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology (currently, independent administrative agency, National
Institute of Technology and Evaluation, International Patent
Organism Depositary, #120, 2-5-8 Kazusakamatari, Kisarazu-shi,
Chiba-ken, 292-0818, Japan) on Feb. 19, 1998, and assigned an
accession number of FERM P-16645. Then, the deposit was converted
into an international deposit under the provisions of the Budapest
Treaty on Jan. 11, 1999, and assigned an accession number of FERM
BP-6616. The SC17sucA strain was assigned a private number of
AJ417, and deposited at the independent administrative agency,
National Institute of Advanced Industrial Science and Technology,
International Patent Organism Depositary (currently, independent
administrative agency, National Institute of Technology and
Evaluation, International Patent Organism Depositary, #120, 2-5-8
Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on Feb.
26, 2004, under an accession number of FERM BP-8646.
The AJ13355 strain was identified as Enterobacter agglomerans when
it was isolated, but it was recently reclassified as Pantoea
ananatis on the basis of nucleotide sequencing of 16S rRNA and so
forth. Therefore, although the AJ13355 and AJ13356 strains are
deposited at the aforementioned depository as Enterobacter
agglomerans, they are referred to as Pantoea ananatis in this
specification.
Furthermore, examples of L-glutamic acid-producing bacteria and
parental strains that can be used to derive such bacteria also
include Pantoea bacteria such as the Pantoea ananatis
SC17sucA/RSFCPG+pSTVCB, Pantoea ananatis AJ13601, Pantoea ananatis
NP106, and Pantoea ananatis NA1. The SC17sucA/RSFCPG+pSTVCB strain
was obtained by introducing the plasmid RSFCPG containing the
citrate synthase gene (gltA), phosphoenolpyruvate carboxylase gene
(ppc), and glutamate dehydrogenase gene (gdhA) derived from
Escherichia coli, and the plasmid pSTVCB containing the citrate
synthase gene (gltA) derived from Brevibacterium lactofermentum,
into the SC17sucA strain. The AJ13601 strain is selected from
SC17sucA/RSFCPG+pSTVCB as a strain resistant to a high
concentration of L-glutamic acid at a low pH. The NP106 strain was
obtained from the AJ13601 strain by curing the RSFCPG and pSTVCB
plasmids. The AJ13601 strain was deposited at the National
Institute of Bioscience and Human Technology, Agency of Industrial
Science and Technology (currently, independent administrative
agency, National Institute of Technology and Evaluation,
International Patent Organism Depositary, #120, 2-5-8
Kazusakamatari, Kisarazu-shi, Chiba-ken, 292-0818, Japan) on Aug.
18, 1999, and assigned an accession number FERM P-17516. Then, the
deposit was converted to an international deposit under the
provisions of the Budapest Treaty on Jul. 6, 2000, and assigned an
accession number FERM BP-7207.
Examples of L-glutamic acid-producing bacteria and parental strains
that can be used to derive such bacteria also include strains in
which both the .alpha.-ketoglutarate dehydrogenase (sucA) activity
and the succinate dehydrogenase (sdh) activity are reduced or
deleted (Japanese Patent Laid-open (Kokai) No. 2010-041920).
Specific examples of such strains include, for example, the
sucAsdhA double-deficient strain of Pantoea ananatis NA1 (Japanese
Patent Laid-open (Kokai) No. 2010-041920).
Examples of L-glutamic acid-producing bacteria and parental strains
that can be used to derive such bacteria also include auxotrophic
mutant strains. Specific examples of auxotrophic mutant strains
include, for example, E. coli VL334thrC.sup.+ (VKPM B-8961, EP
1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and
L-threonine auxotrophic strain having mutations in the thrC and
ilvA genes (U.S. Pat. No. 4,278,765). E. coli VL334thrC.sup.+ is an
L-isoleucine-auxotrophic L-glutamic acid-producing bacterium
obtained by introducing a wild-type allele of the thrC gene into
the VL334 strain. The wild-type allele of the thrC gene was
introduced by the method of general transduction using a
bacteriophage P1 grown on the wild-type E. coli K-12 strain (VKPM
B-7) cells.
Examples of L-glutamic acid-producing bacteria and parental strains
that can be used to derive such bacteria also include strains
having resistance to an aspartic acid analogue. Such strains can
also be deficient in .alpha.-ketoglutarate dehydrogenase activity.
Specific examples of strains having resistance to an aspartic acid
analogue and deficient in the .alpha.-ketoglutarate dehydrogenase
activity include, for example, E. coli AJ13199 (FERM BP-5807, U.S.
Pat. No. 5,908,768), E. coli FFRM P-12379, which additionally has a
lowered L-glutamic acid-decomposing ability (U.S. Pat. No.
5,393,671), and E. coli M13138 (FERM BP-5565, U.S. Pat. No.
6,110,714).
Examples of methods for imparting or enhancing L-glutamic
acid-producing ability also include a method of modifying a
bacterium so that the D-xylulose-5-phosphate phosphoketolase
activity and/or the fructose-6-phosphate phosphoketolase activity
are/is enhanced (Japanese Patent Laid-open (Kohyo) No.
2008-509661). Either one of the D-xylulose-5-phosphate
phosphoketolase activity and the fructose-6-phosphate
phosphoketolase activity may be enhanced, or both may be enhanced.
In this specification, D-xylulose-5-phosphate phosphoketolase and
fructose-6-phosphate phosphoketolase may be collectively referred
to as phosphoketolase.
The D-xylulose-5-phosphate phosphoketolase activity means the
conversion of xylulose-5-phosphate into glycelaldehyde-3-phosphate
and acetyl phosphate while consuming phosphoric acid to release one
molecule of H.sub.2O. This activity can be measured by the method
described by Goldberg, M. et al. (Methods Enzymol., 9, 515-520,
1996) or the method described by L. Meile (J. Bacteriol.,
183:2929-2936, 2001).
The fructose-6-phosphate phosphoketolase activity means conversion
of fructose-6-phosphate into erythrose-4-phosphate and acetyl
phosphate while consuming phosphoric acid to release one molecule
of H.sub.2O. This activity can be measured by the method described
by Racker, E. (Methods Enzymol., 5, 276-280, 1962) or the method
described by L. Meile (J. Bacteriol., 183:2929-2936, 2001).
Examples of methods for imparting or enhancing
L-glutamine-producing ability also include, for example, a method
of enhancing the expression of the yhfK gene (WO2005/085419) or the
ybjL gene (WO2008/133161), which is an L-glutamic acid secretion
gene.
<L-Glutamine-Producing Bacteria>
Examples of methods for imparting or enhancing
L-glutamine-producing ability include, for example, a method of
modifying a bacterium so that the bacterium has an increased
activity or activities of one or more of the L-glutamine
biosynthesis enzymes. Examples of such enzymes include, but are not
particularly limited to, glutamate dehydrogenase (gdhA) and
glutamine synthetase (glnA). The glutamine synthetase activity can
also be enhanced by disruption of the glutamine adenylyltransferase
gene (glnE) or disruption of the PII control protein gene (glnB)
(EP 1229121).
Examples of methods for imparting or enhancing
L-glutamine-producing ability also include, for example, a method
of modifying a bacterium so that the bacterium has a reduced
activity or activities of one or more enzymes that catalyze a
reaction branching away from the biosynthesis pathway of
L-glutamine to generate a compound other than L-glutamine. Examples
of such enzymes include, but are not particularly limited to,
glutaminase.
Examples of L-glutamic acid-producing bacteria and parental strains
that can be used to derive such bacteria include a strain belonging
to the genus Escherichia and having a mutant glutamine synthetase
in which the tyrosine residue of the position 397 is replaced with
another amino acid residue (U.S. Patent Published Application No.
2003/0148474).
<L-Proline-Producing Bacteria>
Examples of methods for imparting or enhancing L-proline-producing
ability include, for example, a method of modifying a bacterium so
that the bacterium has an increased activity or activities of one
or more kinds of enzymes selected from the L-proline biosynthesis
enzymes. Examples of such enzymes include glutamate-5-kinase
(proB), .gamma.-glutamylphosphate reductase, and
pyroline-5-carboxylate reductase (putA). For enhancing the activity
of such an enzyme, for example, the proB gene encoding a glutamate
kinase desensitized to feedback inhibition by L-proline (German
Patent No. 3127361) can be preferably used.
Examples of methods for imparting or enhancing
L-glutamine-producing ability also include, for example, a method
of modifying a bacterium so that the bacterium has a reduced
activity of an enzyme involved in decomposition of L-proline.
Examples of such an enzyme include proline dehydrogenase and
ornithine aminotransferase.
Specific examples of L-proline-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, E. coli NRRL B-12403 and NRRL B-12404 (British Patent No.
2075056), E. coli VKPM B-8012 (Russian Patent Application No.
2000124295), E. coli plasmid mutant strains described in German
Patent No. 3127361, E. coli plasmid mutant strains described by
Bloom F. R. et al. (The 15th Miami winter symposium, 1983, p. 34),
E. coli 702 (VKPM B-8011), which is a 3,4-dehydroxyproline and
azetidine-2-carboxylate resistant strain, and E. coli 702ilvA (VKPM
B-8012), which is an ilvA gene-deficient strain of the 702 strain
(EP 1172433).
<L-Threonine-Producing Bacteria>
Examples of methods for imparting or enhancing
L-threonine-producing ability include, for example, a method of
modifying a bacterium so that the bacterium has an increased
activity or activities of one or more of the L-threonine
biosynthesis enzymes. Examples of such enzymes include, but are not
particularly limited to, aspartokinase III (lysC), aspartate
semialdehyde dehydrogenase (asci), aspartokinase I (thrA),
homoserine kinase (thrB), threonine synthase (thrC), and aspartate
aminotransferase (aspartate transaminase) (aspC). Among these
enzymes, it is preferable to enhance activity or activities of one
or more of aspartokinase III, aspartate semialdehyde dehydrogenase,
aspartokinase I, homoserine kinase, aspartate aminotransferase, and
threonine synthase. Any of the genes encoding the L-threonine
biosynthesis enzymes can be introduced into a bacterium having a
reduced ability to decompose threonine. Examples of such a strain
in which threonine decomposition is suppressed include, for
example, E. coli TDH6, which is deficient in the threonine
dehydrogenase activity (Japanese Patent Laid-open (Kokai) No.
2001-346578).
The activities of the L-threonine biosynthesis enzymes are
inhibited by the endproduct, L-threonine. Therefore, to construct
L-threonine-producing strains, the genes of the L-threonine
biosynthesis enzymes can be modified so that the enzymes are
desensitized to feedback inhibition by L-threonine. The
aforementioned thrA, thrB, and thrC genes constitute the threonine
operon, which forms an attenuator structure. The expression of the
threonine operon is inhibited by isoleucine and threonine in the
culture medium and also suppressed by attenuation. Therefore,
expression of the threonine operon can be enhanced by removing the
leader sequence or the attenuator in the attenuation region (refer
to Lynn, S. P., Burton, W. S., Donohue, T. J., Gould, R. M.,
Gumport, R. L, and Gardner, J. F., J. Mol. Biol. 194:59-69 (1987);
WO02/26993; WO2005/049808; and WO2003/097839).
The native promoter of the threonine operon is located upstream of
the threonine operon, and can be replaced with a non-native
promoter (refer to WO98/04715). Also, the threonine operon may be
constructed so that the threonine biosynthesis genes are expressed
under the control of the repressor and promoter of .lamda.-phage
(European Patent No. 0593792). Furthermore, a bacterium modified so
that it is desensitized to feedback inhibition by L-threonine can
also be obtained by selecting a strain resistant to
.alpha.-amino-.beta.-hydroxyisovaleric acid (AHV), which is an
L-threonine analogue.
The expression amount of the threonine operon that is modified so
as to be desensitized to feedback inhibition by L-threonine as
described above can be increased in a host by increasing its copy
number or by ligating it to a potent promoter. The copy number can
be increased by introducing a plasmid containing the threonine
operon into a host. The copy number can also be increased by
transferring the threonine operon to the genome of a host using a
transposon, Mu-phage, or the like.
Examples of methods for imparting or enhancing
L-threonine-producing ability also include, for example, a method
of imparting L-threonine resistance to a host, and a method of
imparting L-homoserine resistance to a host. Such resistance can be
imparted by, for example, enhancing the expression of a gene that
imparts L-threonine resistance or a gene that imparts L-homoserine
resistance. Examples of the genes that impart the above-mentioned
resistance include rhtA (Res. Microbiol. 154:123-135 (2003)), rhtB
(European Patent Laid-open No. 0994190), rhtC (European Patent
Laid-open No. 1013765), yfiK, and yeaS (European Patent Laid-open
No. 1016710). As for methods for imparting L-threonine resistance
to a host, those described in European Patent Laid-open No. 0994190
and WO90/04636 are exemplary.
Specific examples of L-threonine-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, E. coli TDH-6/pVIC40 (VKPM B-3996, U.S. Pat. Nos.
5,175,107 and 5,705,371), E. coli 472T23/pYN7 (ATCC 98081, U.S.
Pat. No. 5,631,157), E. coli NRRL-21593 (U.S. Pat. No. 5,939,307),
E. coli FERM BP-3756 (U.S. Pat. No. 5,474,918), E. coli FERM
BP-3519 and FERM BP-3520 (U.S. Pat. No. 5,376,538), E. coli MG442
(Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E.
coli VL643 and VL2055 (EP 1149911 A), and E. coli VKPM B-5318 (EP
0593792 B).
The VKPM B-3996 strain is obtained by introducing the plasmid
pVIC40 into the TDH-6 strain. The TDH-6 strain has
sucrose-assimilating ability, is deficient in the thrC gene, and
the ilvA gene has a leaky mutation. This VKPM B-3996 strain also
has a mutation in the rhtA gene, which imparts resistance to high
concentrations of threonine or homoserine. The plasmid pVIC40 is a
plasmid obtained by inserting the thrA*BC operon containing a
mutant thrA gene encoding an aspartokinase-homoserine dehydrogenase
I resistant to feedback inhibition by threonine and the wild-type
thrBC genes into an RSF1010-derived vector (U.S. Pat. No.
5,705,371). This mutant thrA gene encodes an
aspartokinase-homoserine dehydrogenase I which is substantially
desensitized to feedback inhibition by threonine. The B-3996 strain
was deposited on Nov. 19, 1987 at the All-Union Scientific Center
of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia)
under the accession number RIA 1867. This strain was also deposited
at the Russian National Collection of Industrial Microorganisms
(VKPM, FGUP GosNII Genetika, 1 Dorozhny proezd., 1 Moscow 117545,
Russia) on Apr. 7, 1987 under the accession number VKPM B-3996.
The VKPM B-5318 strain is prototrophic with regard to isoleucine,
and harbors the plasmid pPRT614, which corresponds to the plasmid
pVIC40 in which the regulatory region of the threonine operon is
replaced with the temperature-sensitive .lamda.-phage Cl repressor
and PR promoter. The VKPM B-5318 strain was deposited at the
Russian National Collection of Industrial Microorganisms (VKPM,
FGUP GosNII Genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russia)
on May 3, 1990 under the accession number of VKPM B-5318.
The thrA gene, which encodes aspartokinase-homoserine dehydrogenase
I of E. coli, has been elucidated (nucleotide numbers 337 to 2799,
GenBank accession NC_000913.2, gi: 49175990). The thrA gene is
located between the thrL and thrB genes on the chromosome of E.
coli K-12. The thrB gene which encodes homoserine kinase of
Escherichia coli has been elucidated (nucleotide numbers 2801 to
3733, GenBank accession NC_000913.2, gi: 49175990). The thrB gene
is located between the thrA and thrC genes on the chromosome of E.
coli K-12. The thrC gene, which encodes threonine synthase of E.
coli, has been elucidated (nucleotide numbers 3734 to 5020, GenBank
accession NC_000913.2, gi: 49175990). The thrC gene is located
between the thrB gene and the yaaX open reading frame on the
chromosome of E. coli K-12. The thrA*BC operon containing a mutant
thrA gene which encodes an aspartokinase-homoserine dehydrogenase I
resistant to feedback inhibition by threonine and the wild-type
thrBC genes can be obtained from the well-known plasmid pVIC40,
which is present in the threonine-producing strain E. coli VKPM
B-3996 (U.S. Pat. No. 5,705,371).
The rhtA gene of E. coli is located at 18 min on the E. coli
chromosome close to the glnHPQ operon, which encodes components of
the glutamine transport system. The rhtA gene is identical to ORF1
(ybiF gene, nucleotide numbers 764 to 1651, GenBank accession
number AAA218541, gi:440181) and is located between the pexB and
ompX genes. The unit expressing a protein encoded by the ORF1 has
been designated rhtA gene (rht: resistance to homoserine and
threonine). It has also been revealed that the rhtA23 mutation that
imparts resistance to high concentrations of threonine or
homoserine is an A-for-G substitution at position -1 with respect
to the ATG start codon (ABSTRACTS of the 17th International
Congress of Biochemistry and Molecular Biology in conjugation with
Annual Meeting of the American Society for Biochemistry and
Molecular Biology, San Francisco, Calif., Aug. 24-29, 1997,
abstract No. 457; EP 1013765 A).
The asd gene of E. coli has already been elucidated (nucleotide
numbers 3572511 to 3571408, GenBank accession NC_000913.1,
gi:16131307), and can be obtained by PCR (refer to White, T. J., et
al., Trends Genet, 5:185-189, 1989) utilizing primers prepared on
the basis of the nucleotide sequence of the gene. The asd genes of
other microorganisms can also be obtained in a similar manner.
The aspC gene of E. coli has also already been elucidated
(nucleotide numbers 983742 to 984932, GenBank accession
NC_000913.1, gi:16128895), and can be obtained by PCR utilizing
primers prepared on the basis of the nucleotide sequence of the
gene. The aspC genes of other microorganisms can also be obtained
in a similar manner.
<L-Lysine-Producing Bacteria>
Examples of methods for imparting or enhancing L-lysine-producing
ability include, for example, a method of modifying a bacterium so
that the bacterium has an increased activity or activities of the
L-lysine biosynthesis enzymes. Examples of such enzymes include,
but are not particularly limited to, dihydrodipicolinate synthase
(dapA), aspartokinase III (lysC), dihydrodipicolinate reductase
(dapB), diaminopimelate decarboxylase (lysA), diaminopimelate
dehydrogenase (ddh) (U.S. Pat. No. 6,040,160), phosphoenolpyruvate
carboxylase (ppc), aspartate semialdehyde dehydrogenase (asd),
aspartate aminotransferase (aspartate transaminase) (aspC),
diaminopimelate epimerase (dapF), tetrahydrodipicolinate
succinylase (dapD), succinyl diaminopimelate deacylase (dapE), and
aspartase (aspA) (EP 1253195 A). It is preferable to enhance the
activity or activities of one or more of, for example,
dihydrodipicolinate reductase, diaminopimelate decarboxylase,
diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase,
aspartate aminotransferase, diaminopimelate epimerase, aspartate
semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and
succinyl diaminopimelate deacylase. In addition, L-lysine-producing
bacteria and parental strains that can be used to derive such
bacteria can express an increased level of the gene involved in
energy efficiency (cyo) (EP 1170376 A), the gene encoding
nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Pat. No.
5,830,716), the ybjE gene (WO2005/073390), or combinations of
these. Since aspartokinase III (lysC) is subject to feedback
inhibition by L-lysine, a mutant lysC gene encoding an
aspartokinase III desensitized to feedback inhibition by L-lysine
(U.S. Pat. No. 5,932,453) may be used for enhancing the activity of
this enzyme. Furthermore, since dihydrodipicolinate synthase (dapA)
is subject to feedback inhibition by L-lysine, a mutant dapA gene
encoding a dihydrodipicolinate synthase desensitized to feedback
inhibition by L-lysine may be used for enhancing the activity of
this enzyme.
Examples of methods for imparting or enhancing L-lysine-producing
ability also include, for example, a method of modifying a
bacterium so that the bacterium has a reduced activity or
activities of one or more of the enzymes that catalyze a reaction
branching away from the biosynthetic pathway of L-lysine to
generate a compound other than L-lysine. Examples of such enzymes
include, but are not particularly limited to, homoserine
dehydrogenase, lysine decarboxylase (U.S. Pat. No. 5,827,698), and
malic enzyme (WO2005/010175).
Examples of L-lysine-producing bacteria and parental strains that
can be used to derive such bacteria also include mutant strains
having resistance to an L-lysine analogue. L-Lysine analogues
inhibit the growth of bacteria such as bacteria of the family
Enterobacteriaceae and coryneform bacteria, but this inhibition is
fully or partially released when L-lysine is present in the medium.
Examples of these L-lysine analogues include, but are not
particularly limited to, oxalysine, lysine hydroxamate,
S-(2-aminoethyl)-L-cysteine (AEC), .gamma.-methyllysine, and
.alpha.-chlorocaprolactam. Mutant strains having resistance to
these lysine analogues can be obtained by subjecting a bacterium to
a conventional artificial mutagenesis treatment.
Specific examples of L-lysine-producing bacteria and parental
strains that can be used to derive such bacteria include E. coli
AJ11442 (FERM BP-1543, NRRL B-12185, see U.S. Pat. No. 4,346,170)
and E. coli VL611. In these strains, aspartokinase is desensitized
to feedback inhibition by L-lysine.
Specific examples of L-lysine-producing bacteria and parental
strains that can be used to derive such bacteria also include the
E. coli WC196 strain. The WC196 strain was bred by imparting AEC
resistance to the W3110 strain, which was derived from E. coli K-12
(U.S. Pat. No. 5,827,698). The WC196 strain was designated E. coli
AJ13069 and deposited at the National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology
(currently, independent administrative agency, National Institute
of Technology and Evaluation, International Patent Organism
Depositary, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken,
292-0818, Japan) on Dec. 6, 1994 and assigned an accession number
of FERM P-14690. Then, the deposit was converted to an
international deposit under the provisions of the Budapest Treaty
on Sep. 29, 1995, and assigned an accession number of FERM BP-5252
(U.S. Pat. No. 5,827,698).
Preferred examples of L-lysine-producing bacteria include E. coli
WC196.DELTA.cadA.DELTA.ldc and E. coli
WC196.DELTA.cadA.DELTA.ldc/pCABD2 (WO2010/061890). The E. coli
WC196.DELTA.cadA.DELTA.ldc strain is constructed from the WC196
strain by disrupting the cadA and ldcC genes encoding lysine
decarboxylase. The WC196.DELTA.cadA.DELTA.ldc/pCABD2 strain was
constructed by introducing the plasmid pCABD2 containing lysine
biosynthesis enzyme genes (U.S. Pat. No. 6,040,160) into the
WC196.DELTA.cadA.DELTA.ldc strain. The WC196.DELTA.cadA.DELTA.ldc
strain, designated as AJ110692, was deposited at the independent
administrative agency, National Institute of Advanced Industrial
Science and Technology, International Patent Organism Depositary
(currently, independent administrative agency, National Institute
of Technology and Evaluation, International Patent Organism
Depositary, #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken,
292-0818, Japan) on Oct. 7, 2008 as an international deposit, and
assigned an accession number of FERM BP-11027. The plasmid pCABD2
contains a mutant dapA gene derived from Escherichia coli and
encoding a dihydrodipicolinate synthase (DDPS) having a mutation
for desensitization to feedback inhibition by L-lysine, a mutant
lysC gene derived from Escherichia coli and encoding aspartokinase
III having a mutation for desensitization to feedback inhibition by
L-lysine, the dapB gene derived from Escherichia coli and encoding
dihydrodipicolinate reductase, and the ddh gene derived from
Brevibacterium lactofermentum and encoding diaminopimelate
dehydrogenase.
Other examples of L-lysine-producing bacteria also include E. coli
AJIK01 (NITE BP-01520). The AJIK01 strain was designated E. coli
AJ111046, and deposited at the independent administrative agency,
National Institute of Technology and Evaluation, International
Patent Organism Depositary (#122, 2-5-8 Kazusakamatari,
Kisarazu-shi, Chiba-ken, 292-0818, Japan) on Jan. 29, 2013. Then,
it was converted to an international deposit under the provisions
of the Budapest Treaty on May 15, 2014, and assigned an accession
number of NITE BP-01520.
<L-Arginine-Producing Bacteria>
Examples of methods for imparting or enhancing L-arginine-producing
ability include, for example, a method of modifying a bacterium so
that the bacterium has an increased activity or activities of one
or more of the L-arginine biosynthesis enzymes. Examples of such
enzymes include, but are not particularly limited to,
N-acetylglutamate synthase (argA), N-acetylglutamyl phosphate
reductase (argC), ornithine acetyl transferase (argJ),
N-acetylglutamate kinase (argB), acetylornithine transaminase
(argD), acetylornithine deacetylase (argE), ornithine carbamoyl
transferase (argF), argininosuccinate synthetase (argG),
argininosuccinate lyase (argH), and carbamoyl phosphate synthetase
(carAB). As the N-acetylglutamate synthase gene (argA), for
example, a gene encoding a mutant N-acetylglutamate synthase
desensitized to feedback inhibition by L-arginine by substitution
for the amino acid residues corresponding to the positions 15 to 19
of the wild type enzyme (European Patent Laid-open No. 1170361) can
be used.
Specific examples of L-arginine-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, the E. coli 237 strain (VKPM B-7925) (U.S. Patent
Published Application No. 2002/058315A1), derivative strains
introduced with the argA gene encoding a mutant N-acetyl glutamate
synthase (Russian Patent Application No. 2001112869, EP 1170361
A1), E. coli 382 strain derived from the 237 strain and having an
improved acetic acid-assimilating ability (VKPM B-7926, EP 1170358
A1), and E. coli 382ilvA+ strain, which is a strain obtained from
the 382 strain by introducing the wild-type ilvA gene from E. coli
K-12 strain thereto. The E. coli strain 237 was deposited at the
Russian National Collection of Industrial Microorganisms (VKPM,
FGUP GosNII Genetika, 1 Dorozhny proezd., 1 Moscow 117545, Russia)
on Apr. 10, 2000 under an accession number of VKPM B-7925, and the
deposit was converted to an international deposit under the
provisions of Budapest Treaty on May 18, 2001. The E. coli 382
strain was deposited at the Russian National Collection of
Industrial Microorganisms (VKPM, FGUP GosNII Genetika, 1 Dorozhny
proezd., 1 Moscow 117545, Russia) on Apr. 10, 2000 under accession
number of VKPM B-7926.
Examples of L-arginine-producing bacteria and parental strains that
can be used to derive such bacteria also include strains having
resistance to amino acid analogues, and so forth. Examples of such
strains include Escherichia coli mutant strains having resistance
to .alpha.-methylmethionine, p-fluorophenylalanine, D-arginine,
arginine hydroxamate, S-(2-aminoethyl)-cysteine,
.alpha.-methylserine, .beta.-2-thienylalanine, or sulfaguanidine
(refer to Japanese Patent Laid-open (Kokai) No. 56-106598).
<L-Citrulline-Producing Bacteria and L-Ornithine-Producing
Bacteria>
The biosynthetic pathways of L-citrulline and L-ornithine are
common to that of L-arginine. Therefore, an ability to produce
L-citrulline and/or L-ornithine can be imparted or enhanced by
increasing the activity or activities of N-acetylglutamate synthase
(argA), N-acetylglutamyl phosphate reductase (argC), ornithine
acetyltransferase (argJ), N-acetylglutamate kinase (argB),
acetylornithine transaminase (argD), and/or acetylornithine
deacetylase (argE) (WO2006/35831).
<L-Histidine-Producing Bacteria>
Examples of methods for imparting or enhancing
L-histidine-producing ability include, for example, a method of
modifying a bacterium so that the bacterium has an increased
activity or activities of one or more of the L-histidine
biosynthesis enzymes. Examples of such enzymes include, but are not
particularly limited to, ATP phosphoribosyltransferase (hisG),
phosphoribosyl AMP cyclohydrolase (hisI), phosphoribosyl-ATP
pyrophosphohydrolase (hisI),
phosphoribosylformimino-5-aminoimidazole carboxamide ribotide
isomerase (hisA), amidotransferase (hisH), histidinol phosphate
aminotransferase (hisC), histidinol phosphatase (hisB), and
histidinol dehydrogenase (hisD).
Among these enzymes, the L-histidine biosynthesis enzymes encoded
by hisG and hisBHAFI are known to be inhibited by L-histidine.
Therefore, the ability to produce L-histidine can be imparted or
enhanced by, for example, introducing a mutation for conferring
resistance to feedback inhibition into the gene encoding ATP
phosphoribosyltransferase (hisG) (Russian Patent Nos. 2003677 and
2119536).
Specific examples of L-histidine-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, strains belonging to the genus Escherichia, such as the E.
coli 24 strain (VKPM B-5945, RU 2003677), E. coli NRRL B-12116 to
B-12121 (U.S. Pat. No. 4,388,405), E. coli H-9342 (FERM BP-6675)
and H-9343 (FERM BP-6676, U.S. Pat. No. 6,344,347), E. coli H-9341
(FERM BP-6674, EP 1085087), E. coli AI80/pFM201 (U.S. Pat. No.
6,258,554), E. coli FERM P-5038 and FERM P-5048, which have been
introduced with a vector carrying a DNA encoding an
L-histidine-biosynthesis enzyme (Japanese Patent Laid-open (Kokai)
No. 56-005099), E. coli strains introduced with a gene for amino
acid transport (EP 1016710 A), and E. coli 80 strain, which has
been imparted with resistance to sulfaguanidine,
DL-1,2,4-triazole-3-alanine, and streptomycin (VKPM B-7270, Russian
Patent No. 2119536).
<L-Cysteine-Producing Bacteria>
Examples of methods for imparting or enhancing L-cysteine-producing
ability include, for example, a method of modifying a bacterium so
that the bacterium has an increased activity or activities of one
or more of the L-cysteine biosynthesis enzymes. Examples of such
enzymes include, but are not particularly limited to, serine
acetyltransferase (cysE) and 3-phosphoglycerate dehydrogenase
(serA). The serine acetyltransferase activity can be enhanced by,
for example, introducing a mutant cysE gene encoding a mutant
serine acetyltransferase resistant to feedback inhibition by
cysteine into a bacterium. Such a mutant serine acetyltransferase
is disclosed in, for example, Japanese Patent Laid-open (Kokai) No.
11-155571 and U.S. Patent Published Application No. 20050112731.
Furthermore, the 3-phosphoglycerate dehydrogenase activity can be
enhanced by, for example, introducing a mutant serA gene encoding a
mutant 3-phosphoglycerate dehydrogenase resistant to feedback
inhibition by serine into a bacterium. Such a mutant
3-phosphoglycerate dehydrogenase is disclosed in, for example, U.S.
Pat. No. 6,180,373.
Furthermore, examples of methods for imparting or enhancing
L-cysteine-producing ability also include, for example, a method of
modifying a bacterium so that the bacterium has a reduced activity
or activities of one or more enzymes that catalyze a reaction
branching away from the biosynthesis pathway of L-cysteine to
generate a compound other than L-cysteine. Examples of such enzymes
include, for example, enzymes involved in decomposition of
L-cysteine. Examples of the enzymes involved in decomposition of
L-cysteine include, but are not particularly limited to,
cystathionine-.beta.-lyase (metC, Japanese Patent Laid-open (Kokai)
No. 11-155571; Chandra et al., Biochemistry, 21 (1982) 3064-3069),
tryptophanase (tnaA, Japanese Patent Laid-open (Kokai) No.
2003-169668; Austin Newton et al., J. Biol. Chem., 240 (1965)
1211-1218), O-acetylserine sulfhydrylase B (cysM, Japanese Patent
Laid-open (Kokai) No. 2005-245311), the malY gene product (Japanese
Patent Laid-open (Kokai) No. 2005-245311), and the d0191 gene
product of Pantoea ananatis (Japanese Patent Laid-open (Kokai) No.
2009-232844).
Furthermore, examples of methods for imparting or enhancing
L-cysteine-producing ability also include, for example, a method of
enhancing the L-cysteine excretory system, and a method of
enhancing the sulfate/thiosulfate transport system. Examples of
proteins of the L-cysteine excretory system include the protein
encoded by the ydeD gene (Japanese Patent Laid-open (Kokai) No.
2002-233384), the protein encoded by the yfiK gene (Japanese Patent
Laid-open (Kokai) No. 2004-49237), the proteins encoded by the
emrAB, emrKY, yojIH, acrEF, bcr, and cusA genes (Japanese Patent
Laid-open (Kokai) No. 2005-287333), and the protein encoded by the
yeaS gene (Japanese Patent Laid-open (Kokai) No. 2010-187552).
Examples of the proteins of the sulfate/thiosulfate transport
system include the proteins encoded by the cysPTWAM gene
cluster.
Specific examples of L-cysteine-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, E. coli JM15 transformed with different cysE alleles
encoding feedback-resistant serine acetyltransferases (U.S. Pat.
No. 6,218,168, Russian patent application 2003121601), E. coli
W3110 that overexpresses a gene encoding a protein suitable for
secretion of a cytotoxic substance (U.S. Pat. No. 5,972,663), E.
coli strains having a reduced cysteine desulfohydrase activity
(JP11155571A2), and E. coli W3110 having an increased activity of a
positive transcriptional regulator for cysteine regulon encoded by
the cysB gene (WO0127307A1).
<L-Methionine-Producing Bacteria>
Examples of L-methionine-producing bacteria and parental strains
that can be used to derive such bacteria include L-threonine
auxotrophic strains and mutant strains resistant to norleucine
(Japanese Patent Laid-open (Kokai) No. 2000-139471). Examples of
L-methionine-producing bacteria and parental strains that can be
used to derive such bacteria also include a strain containing a
mutant homoserine transsuccinylase resistant to feedback inhibition
by L-methionine (Japanese Patent Laid-open (Kokai) No. 2000-139471,
U.S. Patent Published Application No. 20090029424). Since
L-methionine is biosynthesized via L-cysteine as an intermediate,
L-methionine-producing ability can also be improved by increasing
L-cysteine production (Japanese Patent Laid-open (Kokai) No.
2000-139471, U.S. Patent Published Application No.
20080311632).
Specific examples of L-methionine-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, E. coli AJ11539 (NRRL B-12399), E. coli AJ11540 (NRRL
B-12400), E. coli AJ11541 (NRRL B-12401), E. coli AJ11542 (NRRL
B-12402, British Patent No. 2075055), the E. coli 218 strain (VKPM
B-8125, Russian Patent No. 2209248) and the 73 strain (VKPM B-8126,
Russian Patent No. 2215782), which are resistant to norleucine,
which is an analogue of L-methionine, and E. coli AJ13425
(FERMP-16808, Japanese Patent Laid-open (Kokai) No. 2000-139471).
The AJ13425 strain is an L-threonine auxotrophic strain derived
from the E. coli W3110, in which the methionine repressor is
deleted, the intracellular S-adenosylmethionine synthetase activity
is attenuated, and the intracellular homoserine transsuccinylase
activity, cystathionine .gamma.-synthase activity, and
aspartokinase-homoserine dehydrogenase II activity are
enhanced.
<L-Leucine-Producing Bacteria>
Examples of methods for imparting or enhancing L-leucine-producing
ability include, for example, a method of modifying a bacterium so
that the bacterium has an increased activity or activities of one
or more of the L-leucine biosynthesis enzymes. Examples of such
enzymes include, but are not particularly limited to, the enzymes
encoded by the genes of the leuABCD operon. Furthermore, for
enhancing the activity of such an enzyme, for example, the mutant
leuA gene encoding an isopropyl maleate synthase desensitized to
feedback inhibition by L-leucine (U.S. Pat. No. 6,403,342) can be
used.
Specific examples of L-leucine-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, strains belonging to the genus Escherichia, such as E.
coli strains resistant to leucine (for example, the 57 strain (VKPM
B-7386, U.S. Pat. No. 6,124,121)), E. coli strains resistant to a
leucine analogue such as .beta.-2-thienylalanine, 3-hydroxyleucine,
4-azaleucine, and 5,5,5-trifluoroleucine (Japanese Patent
Publication (Kokoku) No. 62-34397 and Japanese Patent Laid-open
(Kokai) No. 8-70879), E. coli strains obtained by a gene
engineering technique described in WO96/06926, and E. coli H-9068
(Japanese Patent Laid-open (Kokai) No. 8-70879).
<L-Isoleucine-Producing Bacteria>
Examples of methods for imparting or enhancing
L-isoleucine-producing ability include, for example, a method of
modifying a bacterium so that the bacterium has increased activity
or activities of one or more of the L-isoleucine biosynthesis
enzymes. Examples of such enzymes include, but are not particularly
limited to, threonine deaminase and acetohydroxy acid synthase
(Japanese Patent Laid-open (Kokai) No. 2-458, FR 0356739, U.S. Pat.
No. 5,998,178).
Specific examples of L-isoleucine-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, Escherichia bacteria such as mutant strains having
resistance to 6-dimethylaminopurine (Japanese Patent Laid-open
(Kokai) No. 5-304969), mutant strains having resistance to an
isoleucine analogue such as thiaisoleucine and isoleucine
hydroxamate, and mutant strains having resistance to such an
isoleucine analogue and further having resistance to DL-ethionine
and/or arginine hydroxamate (Japanese Patent Laid-open (Kokai) No.
5-130882).
<L-Valine-Producing Bacteria>
Examples of methods for imparting or enhancing L-valine-producing
ability include, for example, a method of modifying a bacterium so
that the bacterium has an increased activity or activities of one
or more of the L-valine biosynthesis enzymes. Examples of such
enzymes include, but are not particularly limited to, the enzymes
encoded by the genes of the ilvGMEDA operon and the enzymes encoded
by the ilvBNC operon. The ilvBN gene encodes acetohydroxy acid
synthase, and the ilvC gene encodes isomeroreductase (WO00/50624).
Expressions of the ilvGMEDA operon and the ilvBNC operon are
suppressed (attenuated) by L-valine, L-isoleucine, and/or
L-leucine. Therefore, to enhance the activity of such an enzyme,
the suppression of expression by the produced L-valine can be
released by removing or modifying a region required for the
attenuation. Furthermore, the threonine deaminase encoded by the
ilvA gene is an enzyme that catalyzes the deamination reaction of
L-threonine resulting 2-ketobutyric acid, which is the
rate-limiting step of the L-isoleucine biosynthesis system.
Therefore, for L-valine production, the ilvA gene can be, for
example, disrupted, and thereby the threonine deaminase activity is
decreased.
Examples of methods for imparting or enhancing L-valine-producing
ability also include, for example, a method of modifying a
bacterium so that the bacterium has a reduced activity or
activities of one or more enzymes that catalyze a reaction
branching away from the biosynthesis pathway of L-valine to
generate a compound other than L-valine. Examples of such enzymes
include, but are not particularly limited to, threonine dehydratase
involved in the L-leucine synthesis, and the enzymes involved in
the D-pantothenic acid synthesis (WO00/50624).
Specific examples of L-valine-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, E. coli strains modified so as to overexpress the ilvGMEDA
operon (U.S. Pat. No. 5,998,178).
Examples of L-valine-producing bacteria and parental strains that
can be used to derive such bacteria also include mutant strains
having a mutation in amino-acyl t-RNA synthetase (U.S. Pat. No.
5,658,766). Examples of such strains include, for example, E. coli
VL1970, which has a mutation in the ileS gene encoding isoleucine
t-RNA synthetase. E. coli VL1970 was deposited at the Russian
National Collection of Industrial Microorganisms (VKPM, FGUP GosNII
Genetika, 1 Dorozhny Proezd, 1 Moscow 117545, Russia) on Jun. 24,
1988 under the accession number of VKPM B-4411. Examples of
L-valine-producing bacteria and parental strains that can be used
to derive such bacteria also include mutant strains requiring
lipoic acid for growth and/or lacking H.sup.+-ATPase
(WO96/06926).
<L-Tryptophan-Producing Bacteria, L-Phenylalanine-Producing
Bacteria, and L-Tyrosine-Producing Bacteria>
Examples of methods for imparting or enhancing
L-tryptophan-producing ability, L-phenylalanine-producing ability,
and/or L-tyrosine-producing ability include, for example, a method
of modifying a bacterium so that the bacterium has an increased
activity or activities of one or more of the L-tryptophan,
L-phenylalanine, and/or L-tyrosine biosynthesis enzymes.
Examples of enzymes having common biosynthesis systems of these
aromatic amino acids include, but not particularly limited to,
3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG),
3-dehydroquinate synthase (aroB), shikimate dehydrogenase (aroE),
shikimate kinase (aroL), 5-enolpyruvylshikimate-3-phosphate
synthase (aroA), and chorismate synthase (aroC) (European Patent
No. 763127). The expressions of the genes encoding these enzymes
are controlled by the tyrosine repressor (tyrR), and the activities
of these enzymes may be enhanced by deleting the tyrR gene
(European Patent No. 763127).
Examples of the L-tryptophan biosynthesis enzymes include, but are
not particularly limited to, anthranilate synthase (trpE),
tryptophan synthase (trpAB), and phosphoglycerate dehydrogenase
(serA). For example, by introducing a DNA containing the tryptophan
operon, L-tryptophan-producing ability can be imparted or enhanced.
Tryptophan synthase consists of .alpha. and .beta. subunits encoded
by the trpA and trpB genes, respectively. Since the anthranilate
synthase is subject to feedback inhibition by L-tryptophan, a gene
encoding this enzyme introduced with a mutation for desensitization
to feedback inhibition may be used for enhancing the activity of
that enzyme. Since the phosphoglycerate dehydrogenase is subject to
feedback inhibition by L-serine, a gene encoding this enzyme
introduced with a mutation for desensitization to feedback
inhibition may be used for enhancing the activity of that enzyme.
Furthermore, by increasing the expression of the operon (ace
operon) consisting of the maleate synthase gene (aceB), isocitrate
lyase gene (aceA), and isocitrate dehydrogenase kinase/phosphatase
gene (aceK), L-tryptophan-producing ability may be imparted or
enhanced (WO2005/103275).
Examples of the L-phenylalanine biosynthesis enzymes include, but
are not particularly limited to, chorismate mutase and prephenate
dehydratase. The chorismate mutase and prephenate dehydratase are
encoded by the pheA gene as a bifunctional enzyme. Since the
chorismate mutase and prephenate dehydratase are subject to
feedback inhibition by L-phenylalanine, genes encoding these
enzymes introduced with a mutation for desensitization to feedback
inhibition may be used to enhance the activities of these
enzymes.
Examples of the L-tyrosine biosynthesis enzymes include, but are
not particularly limited to, chorismate mutase and prephenate
dehydrogenase. The chorismate mutase and prephenate dehydrogenase
are encoded by the tyrA gene as a bifunctional enzyme. Since the
chorismate mutase and prephenate dehydrogenase are subject to
feedback inhibition by L-tyrosine, genes encoding these enzymes
introduced with a mutation for desensitization to feedback
inhibition may be used to enhance the activities of these
enzymes.
The L-tryptophan, L-phenylalanine, and/or L-tyrosine-producing
bacteria may be modified so that biosynthesis of an aromatic amino
acid other than the objective aromatic amino acid is reduced.
Furthermore, the L-tryptophan, L-phenylalanine, and/or
L-tyrosine-producing bacteria may be modified so that a by-product
uptake system is enhanced. Examples of the by-product include
aromatic amino acids other than the objective aromatic amino acid.
Examples of the gene encoding such a by-product uptake system
include, for example, tnaB and mtr, which are genes encoding the
L-tryptophan uptake system, pheP, which is a gene encoding the
L-phenylalanine uptake system, and tyrP, which is a gene encoding
the L-tyrosine uptake system (EP 1484410).
Specific examples of L-tryptophan-producing bacteria and parental
strains that can be used to derive such bacteria include, for
example, E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91
(DSM10123), which have a mutant trpS gene encoding a partially
inactivated tryptophanyl-tRNA synthetase (U.S. Pat. No. 5,756,345),
E. coli SV164, which has a trpE allele encoding an anthranilate
synthase desensitized to feedback inhibition by tryptophan, E. coli
SV164 (pGH5), which has a serA allele encoding a phosphoglycerate
dehydrogenase desensitized to feedback inhibition by serine and a
trpE allele encoding an anthranilate synthase desensitized to
feedback inhibition by tryptophan (U.S. Pat. No. 6,180,373), a
strain introduced with a tryptophan operon containing a trpE allele
encoding an anthranilate synthase desensitized to feedback
inhibition by tryptophan (Japanese Patent Laid-open (Kokai) Nos.
57-71397 and 62-244382, U.S. Pat. No. 4,371,614), E. coli
AGX17(pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264),
which are deficient tryptophanase (U.S. Pat. No. 4,371,614), E.
coli AGX17/pGX50, pACKG4-pps, which has an increased
phosphoenolpyruvate-producing ability (WO97/08333, U.S. Pat. No.
6,319,696), and strains belonging to the genus Escherichia having
an increased activity of the protein encoded by the yedA or yddG
gene (U.S. Patent Published Applications 2003/0148473 A1 and
2003/0157667 A1).
Specific examples of L-phenylalanine-producing bacteria and
parental strains that can be used to derive such bacteria include,
for example, E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197),
which is deficient in the chorismate mutase-prephenate
dehydrogenase and the tyrosine repressor (WO03/044191), E. coli
HW1089 (ATCC 55371), which contains a mutant pheA34 gene encoding a
chorismate mutase-prephenate dehydratase desensitized to feedback
inhibition (U.S. Pat. No. 5,354,672), E. coli MWEC101-b
(KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146, and
NRRL B-12147 (U.S. Pat. No. 4,407,952). Specific examples of
L-phenylalanine-producing bacteria and parental strains that can be
used to derive such bacteria also include, for example, E. coli
K-12<W3110(tyrA)/pPHAB> (FERM BP-3566), E. coli
K-12<W3110(tyrA)/pPHAD> (FERM BP-12659), E. coli
K-12<W3110(tyrA)/pPHATerm> (FERM BP-12662), and E. coli K-12
AJ12604<W3110(tyrA)/pBR-aroG4, pACMAB> (FERM BP-3579), which
contains a gene encoding a chorismate mutase-prephenate dehydratase
desensitized to feedback inhibition (EP 488424 B1). Specific
examples of L-phenylalanine-producing bacteria and parental strains
that can be used to derive such bacteria further include, for
example, strains belonging to the genus Escherichia having an
increased activity of the protein encoded by the yedA gene or the
yddG gene (U.S. Patent Published Applications Nos. 2003/0148473 and
2003/0157667, WO03/044192).
Furthermore, examples of methods for imparting or enhancing an
L-amino acid-producing ability include, for example, a method of
modifying a bacterium so that the bacterium has an increased
activity for secreting an L-amino acid from a bacterial cell. Such
an activity for secreting an L-amino acid can be increased by, for
example, increasing the expression of a gene encoding a protein
responsible for secretion of the L-amino acid. Examples of genes
encoding the proteins responsible for secretion of various amino
acids include, for example, b2682 (ygaZ), b2683 (ygaH), b1242
(ychE), and b3434 (yhgN) (Japanese Patent Laid-open (Kokai) No.
2002-300874).
Furthermore, examples of methods for imparting or enhancing an
L-amino acid-producing ability also include, for example, a method
of modifying a bacterium so that the bacterium has an increased
activity or activities of one or more of the proteins involved in
the glycometabolism and energy metabolism.
Examples of the proteins involved in the glycometabolism include
proteins involved in uptake of saccharides and the glycolysis
system enzymes. Examples of genes encoding a protein involved in
glycometabolism include genes encoding glucose-6-phosphate
isomerase (pgi, WO01/02542), pyruvate carboxylase (pyc, WO99/18228,
European Patent Laid-open No. 1092776), phosphoglucomutase (pgm,
WO03/04598), fructose bisphosphate aldolase (pfkB, fbp,
WO03/04664), transaldolase (talB, WO03/008611), fumarase (fum,
WO01/02545), non-PTS sucrose uptake (csc, European Patent Laid-open
No. 149911), and sucrose assimilation (scrAB operon,
WO90/04636).
Examples of genes encoding the proteins involved in the energy
metabolism include the transhydrogenase gene (pntAB, U.S. Pat. No.
5,830,716) and cytochrome bo-type oxidase gene (cyoB, European
Patent Laid-open No. 1070376).
The genes used for the breeding of the aforementioned L-amino
acid-producing bacteria are not limited to the genes exemplified
above and genes having a known nucleotide sequence, and may include
variants of these genes, so long as the original function of the
gene is maintained. For example, the genes used for the breeding of
the L-amino acid-producing bacteria may be encode a protein having
an amino acid sequence of a known protein, but include
substitution, deletion, insertion, or addition of one or several
amino acid residues at one or several positions. For the variants
of genes and proteins, the descriptions concerning variants of
aconitase and acetaldehyde dehydrogenase, and genes encoding them
mentioned later can be similarly applied.
<1-2> Enhancement of Aconitase Activity and Acetaldehyde
Dehydrogenase Activity
The bacterium has been modified so that the activity of aconitase
is increased, or both the activities of aconitase and acetaldehyde
dehydrogenase are increased. By modifying a bacterium so that the
activity of aconitase is increased, or both the activities of
aconitase and acetaldehyde dehydrogenase are increased, L-amino
acid production by the bacterium using ethanol as a carbon source
can be improved.
The bacterium can be obtained by modifying a bacterium having an
L-amino acid-producing ability so that the activity of aconitase is
increased, or both the activities of aconitase and acetaldehyde
dehydrogenase are increased. Furthermore, the bacterium can also be
obtained by modifying a bacterium so that the activity of aconitase
is increased, or the activities of aconitase and acetaldehyde
dehydrogenase are increased, and then imparting an L-amino acid
producing ability to the bacterium or enhancing L-amino
acid-producing ability of the bacterium. The bacterium may also
acquire an L-amino acid-producing ability by being modified so that
the activity of aconitase is increased, or both the activities of
aconitase and acetaldehyde dehydrogenase are increased. The
modifications for constructing the bacterium can be performed in an
arbitrary order.
The term "aconitase" refers to a protein having an activity for
reversibly catalyzing the isomerization between citrate and
isocitrate (EC 4.2.1.3). This activity is also referred to as
"aconitase activity". A gene that encodes aconitase is also
referred to as "aconitase gene". The aconitase activity can be
measured by, for example, measuring generation of cis-aconitate
from isocitrate (Gruer M J, Guest J R., Microbiology., 1994,
October; 140 (10):2531-41).
Examples of aconitase include the AcnB protein, which is encoded by
the acnB gene, and the AcnA protein, which is encoded by the acnA
gene. For example, the activity of the AcnA protein may be
enhanced, the activity of the AcnB protein may be enhanced, or the
activities of both the AcnA protein and AcnB protein may be
enhanced. When the bacterium has not been modified so that the
activity of acetaldehyde dehydrogenase is increased, at least the
activity of the AcnB protein is enhanced.
Examples of the AcnA protein and AcnB protein include, for example,
AcnA proteins and AcnB proteins of bacteria belonging to the family
Enterobacteriaceae such as Escherichia coli, Pantoea ananatis,
Pectobacterium atrosepticum (formerly, Envinia carotovora), and
Salmonella enterica.
The acnA gene of the Escherichia coli K-12 MG1655 strain
corresponds to the sequence of the positions 1335831 to 1338506 in
the genome sequence registered at the NCBI database as GenBank
accession NC_000913 (VERSION NC_000913.3 GI: 556503834). The AcnA
protein of the MG1655 strain is registered as GenBank accession
NP_415792 (version NP_415792.1 GI: 16129237). The nucleotide
sequence of the acnA gene and the amino acid sequence of the AcnA
protein of the MG1655 strain are shown as SEQ ID NOS: 21 and 22,
respectively.
The acnA gene of the Pantoea ananatis AJ13355 strain corresponds to
the complementary sequence of the sequence of the positions 1665681
to 1668362 in the genome sequence registered at the NCBI database
as GenBank accession NC_017531 (VERSION NC_017531.1 GI: 386014600).
The AcnA protein of the AJ13355 strain is registered as GenBank
accession YP_005934253 (version YP_005934253.1 GI: 386015968). The
nucleotide sequence of the acnA gene and the amino acid sequence of
the AcnA protein of the AJ13355 strain are shown as SEQ ID NOS: 23
and 24, respectively.
The acnA gene of the Pectobacterium atrosepticum SCRI1043 strain
corresponds to the sequence of the positions 2198282 to 2200954 in
the genome sequence registered at the NCBI database as GenBank
accession NC_004547 (VERSION NC_004547.2 GI: 50119055). The AcnA
protein of the SCRI1043 strain is registered as GenBank accession
YP_050038 (version YP_050038.1 GI: 50120871). The nucleotide
sequence of the acnA gene and the amino acid sequence of the AcnA
protein of the SCRI1043 strain are shown as SEQ ID NOS: 25 and 26,
respectively.
The acnA gene of the Salmonella enterica serovar Typhi CT18 strain
corresponds to the sequence of the positions 1298278 to 1300953 in
the genome sequence registered at the NCBI database as GenBank
accession NC_003198 (VERSION NC_003198.1 GI: 16762629). The AcnA
protein of the CT18 strain is registered as GenBank accession
NP_455785 (version NP_455785.1 GI: 16760168). The nucleotide
sequence of the acnA gene and the amino acid sequence of the AcnA
protein of the CT18 strain are shown as SEQ ID NOS: 27 and 28,
respectively.
The acnB gene of the Escherichia coli K-12 MG1655 strain
corresponds to the sequence of the positions 131615 to 134212 in
the genome sequence registered at the NCBI database as GenBank
accession NC_000913 (VERSION NC_000913.3 GI: 556503834). The AcnB
protein of the MG1655 strain is registered as GenBank accession
NP_414660 (version NP_414660.1 GI: 16128111). The nucleotide
sequence of the acnB gene and the amino acid sequence of the AcnB
protein of the MG1655 strain are shown as SEQ ID NOS: 29 and 30,
respectively.
The acnB gene of the Pantoea ananatis AJ13355 strain corresponds to
the sequence of the positions 116856 to 119552 in the genome
sequence registered at the NCBI database as GenBank accession NC
017531 (VERSION NC 017531.1 GI: 386014600). The AcnB protein of the
AJ13355 strain is registered as GenBank accession YP_005932972
(version YP_005932972.1 GI: 386014695). The nucleotide sequence of
the acnB gene and the amino acid sequence of the AcnB protein of
the AJ13355 strain are shown as SEQ ID NOS: 31 and 32,
respectively.
The acnB gene of the Pectobacterium atrosepticum SCRI1043 strain
corresponds to the complementary sequence of the sequence of the
positions 4218908 to 4221505 in the genome sequence registered at
the NCBI database as GenBank accession NC 004547 (VERSION
NC_004547.2 GI: 50119055). The AcnB protein of the SCRI1043 strain
is registered as GenBank accession YP_051867 (version YP_051867.1
GI: 50122700). The nucleotide sequence of the acnB gene and the
amino acid sequence of the AcnB protein of the SCRI1043 strain are
shown as SEQ ID NOS: 33 and 34, respectively.
The acnB gene of the Salmonella enterica serovar Typhi CT18 strain
corresponds to the sequence of the positions 189006 to 191603 in
the genome sequence registered at the NCBI database as GenBank
accession NC_003198 (VERSION NC_003198.1 GI: 16762629). The AcnB
protein of the CT18 strain is registered as GenBank accession
NP_454772 (version NP_454772.1 GI: 16759155). The nucleotide
sequence of the acnB gene and the amino acid sequence of the AcnB
protein of the CT18 strain are shown as SEQ ID NOS: 35 and 36,
respectively.
The term "acetaldehyde dehydrogenase" refers to a protein that
reversibly catalyzes the reaction of generating acetic acid from
acetaldehyde by using NAD.sup.+ or NADP.sup.+ as an electron
acceptor (EC 1.2.1.3, EC 1.2.1.4, EC 1.2.1.5, EC 1.2.1.22, etc.).
This activity is also referred to as "acetaldehyde dehydrogenase
activity". A gene encoding acetaldehyde dehydrogenase is also
referred to as "acetaldehyde dehydrogenase gene". The acetaldehyde
dehydrogenase activity can be measured by, for example, measuring
acetaldehyde-dependent reduction of NAD.sup.+ or NADP.sup.+ (Ho K
K, Weiner H., J. Bacteriol., 2005, February; 187(3): 1067-73).
Acetaldehyde dehydrogenase is also referred to as "CoA-independent
acetaldehyde dehydrogenase", and is distinguished from
CoA-dependent acetaldehyde dehydrogenase (to be explained later).
Acetaldehyde dehydrogenase is also referred to as "aldehyde
dehydrogenase", "lactaldehyde dehydrogenase", or the like.
Examples of the acetaldehyde dehydrogenase include A1 dB protein,
which is encoded by aldB gene. Examples of the A1 dB protein
include, for example, A1 dB proteins of bacteria belonging to the
family Enterobacteriaceae such as Escherichia coli, Pantoea
ananatis, Pectobacterium atrosepticum (formerly, Envinia
carotovora), and Salmonella enterica.
The aldB gene of the Escherichia coli K-12 MG1655 strain
corresponds to the complementary sequence of the sequence of the
positions 3754973 to 3756511 in the genome sequence registered at
the NCBI database as GenBank accession NC_000913 (VERSION
NC_000913.3 GI: 556503834). The A1 dB protein of the MG1655 strain
is registered as GenBank accession NP_418045 (version NP_418045.4
GI: 90111619). The nucleotide sequence of the aldB gene and the
amino acid sequence of the A1 dB protein of the MG1655 strain are
shown as SEQ ID NOS: 37 and 38, respectively.
An aldB gene homologue of the Pantoea ananatis LMG 20103 strain is
registered as one of aldA genes at a database. This aldB gene
homologue is regarded as the aldB gene. The aldB gene of the
Pantoea ananatis LMG 20103 strain corresponds to the complementary
sequence of the sequence of the positions 2168098 to 2169570 in the
genome sequence registered at the NCBI database as GenBank
accession NC_013956 (VERSION NC_013956.2 GI: 332139403). The AldB
protein of the LMG 20103 strain is registered as GenBank accession
YP_003520235 (version YP_003520235.1 GI: 291617493). The nucleotide
sequence of the aldB gene and the amino acid sequence of the A1 dB
protein of the LMG 20103 strain are shown as SEQ ID NOS: 39 and 40,
respectively.
The aldB gene of the Pectobacterium atrosepticum SCRI1043 strain
corresponds to the sequence of the positions 111626 to 113161 in
the genome sequence registered at the NCBI database as GenBank
accession NC_004547 (VERSION NC_004547.2 GI: 50119055). The A1 dB
protein of the SCRI1043 strain is registered as GenBank accession
YP_048222 (version YP_048222.1 GI: 50119055). The nucleotide
sequence of the aldB gene and the amino acid sequence of the A1 dB
protein of the SCRI1043 strain are shown as SEQ ID NOS: 41 and 42,
respectively.
The aldB gene of the Salmonella enterica serovar Typhi CT18 strain
corresponds to the sequence of the positions 3978586 to 3980124 in
the genome sequence registered at the NCBI database as GenBank
accession NC_003198 (VERSION NC_003198.1 GI: 16762629). The A1 dB
protein of the CT18 strain is registered as GenBank accession
NP_458246 (version NP_458246.1 GI: 16762629). The nucleotide
sequence of the aldB gene and the amino acid sequence of the A1 dB
protein of the CT18 strain are shown as SEQ ID NOS: 43 and 44,
respectively.
The result of alignment of these AldB proteins is shown in FIG.
1A-1B. The homologies of the amino acid sequence of the AldB
protein of the Escherichia coli K-12 MG1655 strain to the amino
acid sequences of the AldB proteins of the Pantoea ananatis LMG
20103 strain, Pectobacterium atrosepticum SCRI1043 strain, and
Salmonella enterica serovar Typhi CT18 strain are 64.7%, 81.4%, and
95.8%, respectively.
That is, the aconitase gene may be, for example, a gene having the
nucleotide sequence shown as SEQ ID NO: 21, 23, 25, 27, 29, 31, 33,
or 35. Also, aconitase may be, for example, a protein having the
amino acid sequence shown as SEQ ID NO: 22, 24, 26, 28, 30, 32, 34,
or 36. Also, the acetaldehyde dehydrogenase gene may be, for
example, a gene having the nucleotide sequence shown as SEQ ID NO:
37, 39, 41, or 43. Also, acetaldehyde dehydrogenase may be, for
example, a protein having the amino acid sequence shown as SEQ ID
NO: 38, 40, 42, or 44. The expression "a gene or protein has a
nucleotide or amino acid sequence" encompasses cases where a gene
or protein comprises the nucleotide or amino acid sequence, and
cases where a gene or protein consists of the nucleotide or amino
acid sequence.
Aconitase may be a variant of any of the aconitases exemplified
above (e.g. AcnA proteins and AcnB proteins exemplified above), so
long as the original function thereof is maintained. Similarly, the
aconitase gene may be a variant of any of the aconitase genes
exemplified above (e.g. acnA genes and acnB genes exemplified
above), so long as the original function thereof is maintained.
Also, acetaldehyde dehydrogenase may be a variant of any of the
acetaldehyde dehydrogenases exemplified above (e.g. AldB proteins
exemplified above), so long as the original function thereof is
maintained. Similarly, the acetaldehyde dehydrogenase gene may be a
variant of any of the acetaldehyde dehydrogenase genes exemplified
above (e.g. aldB genes exemplified above), so long as the original
function is maintained. Such a variant that maintains the original
function is also referred to as a "conservative variant". Examples
of the conservative variants include, for example, homologues and
artificially modified versions of the aconitases and acetaldehyde
dehydrogenases exemplified above and genes encoding them.
The terms "AcnA protein", "AcnB protein", and "AldB protein",
include not only the AcnA proteins, AcnB proteins, and AldB
proteins exemplified above, respectively, but also includes
respective conservative variants thereof. Similarly, the terms
"acnA gene", "acnB gene", and "aldB gene" include not only the acnA
genes, acnB genes, and aldB genes exemplified above, but also
includes respective conservative variants thereof.
The expression "the original function is maintained" means that a
variant of gene or protein has a function (such as activity or
property) corresponding to the function (such as activity or
property) of the original gene or protein. That is, the expression
"the original function is maintained" used for aconitase means that
a variant of the protein has the aconitase activity, and the
expression "the original function is maintained" used for
acetaldehyde dehydrogenase means that a variant of the protein has
the acetaldehyde dehydrogenase activity. Also, the expression "the
original function is maintained" used for the aconitase gene means
that a variant of the gene encodes a protein of which the original
function is maintained, i.e. a protein having the aconitase
activity, and the expression "the original function is maintained"
used for the acetaldehyde dehydrogenase gene means that a variant
of the gene encodes a protein of which the original function is
maintained, i.e. a protein having the acetaldehyde dehydrogenase
activity.
Hereafter, examples of the conservative variants will be
explained.
Examples of homologues of aconitase and acetaldehyde dehydrogenase
include, for example, proteins that can be obtained from public
databases by BLAST search or FASTA search using any of the
aforementioned amino acid sequences as a query sequence.
Furthermore, homologues of the aconitase and acetaldehyde
dehydrogenase genes can be obtained by, for example, PCR using a
chromosome of various microorganisms as the template, and
oligonucleotides prepared on the basis of any of the aforementioned
nucleotide sequences as primers.
Aconitase or acetaldehyde dehydrogenase may be a protein having any
of the aforementioned amino acid sequences, that is, the amino acid
sequence shown as SEQ ID NO: 24, 26, 28, 30, 32, 34, or 36 for
aconitase, or the amino acid sequence shown as SEQ ID NO: 38, 40,
42, or 44 for acetaldehyde dehydrogenase, but which includes
substitution, deletion, insertion, or addition of one or several
amino acid residues at one or several positions, so long as the
original function is maintained. Although the number meant by the
term "one or several" mentioned above may differ depending on the
positions of amino acid residues in the three-dimensional structure
of the protein or the types of amino acid residues, specifically,
it is, for example, 1 to 50, 1 to 40, or 1 to 30, 1 to 20, 1 to 10,
1 to 5, or 1 to 3.
The aforementioned substitution, deletion, insertion, or addition
of one or several amino acid residues is a conservative mutation
that maintains the normal function of the protein. Typical examples
of the conservative mutation are conservative substitutions. The
conservative substitution is a mutation wherein substitution takes
place mutually among Phe, Trp, and Tyr, if the substitution site is
an aromatic amino acid; among Leu, Ile, and Val, if it is a
hydrophobic amino acid; between Gln and Asn, if it is a polar amino
acid; among Lys, Arg, and His, if it is a basic amino acid; between
Asp and Glu, if it is an acidic amino acid; and between Ser and
Thr, if it is an amino acid having a hydroxyl group. Examples of
substitutions considered as conservative substitutions include,
specifically, substitution of Ser or Thr for Ala, substitution of
Gln, His, or Lys for Arg, substitution of Glu, Gln, Lys, His, or
Asp for Asn, substitution of Asn, Glu, or Gln for Asp, substitution
of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp, or
Arg for Gln, substitution of Gly, Asn, Gln, Lys, or Asp for Glu,
substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg, or
Tyr for His, substitution of Leu, Met, Val, or Phe for Ile,
substitution of Ile, Met, Val, or Phe for Leu, substitution of Asn,
Glu, Gln, His, or Arg for Lys, substitution of Ile, Leu, Val, or
Phe for Met, substitution of Trp, Tyr, Met, Ile, or Leu for Phe,
substitution of Thr or Ala for Ser, substitution of Ser or Ala for
Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe,
or Trp for Tyr, and substitution of Met, Ile, or Leu for Val.
Furthermore, such substitution, deletion, insertion, addition,
inversion, or the like of amino acid residues as mentioned above
includes a naturally occurring mutation due to an individual
difference, or a difference of species of the organism from which
the protein is derived (mutant or variant).
Aconitase or acetaldehyde dehydrogenase may be a protein having an
amino acid sequence showing a homology of, for example, 80% or
more, 90% or more, 95% or more, 97% or more, 99% or more, to the
total amino acid sequence of any of the aforementioned amino acid
sequences, so long as the original function is maintained. In this
description, "homology" can mean "identity".
Aconitase or acetaldehyde dehydrogenase may be a protein encoded by
a DNA that is able to hybridize under stringent conditions with a
probe that can be prepared from any of the aforementioned
nucleotide sequences, that is, the nucleotide sequence shown as SEQ
ID NO: 21, 23, 25, 27, 29, 31, 33, or 35 for aconitase, or the
nucleotide sequence shown as SEQ ID NO: 37, 39, 41, or 43 for
acetaldehyde dehydrogenase, such as a sequence complementary to a
partial or entire sequence of any of the aforementioned nucleotide
sequences, so long as the original function is maintained. The
"stringent conditions" refer to conditions under which a so-called
specific hybrid is formed, and a non-specific hybrid is not formed.
Examples of the stringent conditions include those under which
highly homologous DNAs hybridize to each other, for example, DNAs
not less than 80% homologous, not less than 90% homologous, not
less than 95% homologous, not less than 97% homologous, or not less
than 99% homologous, hybridize to each other, and DNAs less
homologous than the above do not hybridize to each other, or
conditions of washing of typical Southern hybridization, that is,
conditions of washing once, or 2 or 3 times, at a salt
concentration and temperature corresponding to 1.times.SSC, 0.1%
SDS at 60.degree. C., 0.1.times.SSC, 0.1% SDS at 60.degree. C., or
0.1.times.SSC, 0.1% SDS at 68.degree. C. Furthermore, for example,
when a DNA fragment having a length of about 300 bp is used as the
probe, the washing conditions of the hybridization may be, for
example, 50.degree. C., 2.times.SSC and 0.1% SDS.
The percentage of the sequence identity between two sequences can
be determined by, for example, using a mathematical algorithm.
Non-limiting examples of such a mathematical algorithm include the
algorithm of Myers and Miller (1988) CABIOS 4:11-17, the local
homology algorithm of Smith et al (1981) Adv. Appl. Math. 2:482,
the homology alignment algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443-453, the method for searching homology of Pearson
and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and a
modified version of the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 87:2264, such as that described in
Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA
90:5873-5877.
By using a program based on such a mathematical algorithm, sequence
comparison, for example, alignment, for determining the sequence
identity can be performed. The program can be appropriately
executed by a computer. Examples of such a program include, but not
limited to, CLUSTAL of PC/Gene program (available from
Intelligenetics, Mountain View, Calif.), ALIGN program (Version
2.0), and GAP, BESTFIT, BLAST, FASTA, and TFASTA of Wisconsin
Genetics Software Package, Version 8 (available from Genetics
Computer Group (GCG), 575 Science Drive, Madison, Wis., USA).
Alignment using these programs can be performed by using, for
example, initial parameters. The CLUSTAL program is well described
in Higgins et al. (1988) Gene 73:237-244 (1988), Higgins et al.
(1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res.
16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et
al. (1994) Meth. Mol. Biol. 24:307-331.
In order to obtain a nucleotide sequence homologous to a target
nucleotide sequence, in particular, for example, BLAST nucleotide
search can be performed by using BLASTN program with score of 100
and word length of 12. In order to obtain an amino acid sequence
homologous to a target protein, in particular, for example, BLAST
protein search can be performed by using BLASTX program with score
of 50 and word length of 3. See ncbi.nlm.nih.gov for BLAST
nucleotide search and BLAST protein search. In addition, Gapped
BLAST (BLAST 2.0) can be used in order to obtain an alignment
including gap(s) for the purpose of comparison. In addition,
PSI-BLAST can be used in order to perform repetitive search for
detecting distant relationships between sequences. See Altschul et
al. (1997) Nucleic Acids Res. 25:3389 for Gapped BLAST and
PSI-BLAST. When using BLAST, Gapped BLAST, or PSI-BLAST, initial
parameters of each program (e.g. BLASTN for nucleotide sequences,
and BLASTX for amino acid sequences) can be used. Alignment can
also be manually performed.
The sequence identity between two sequences is calculated as the
ratio of residues matching in the two sequences when aligning the
two sequences so as to fit maximally with each other.
Furthermore, since the degeneracy of codons differs depending on
the host, arbitrary codons in the aconitase or acetaldehyde
dehydrogenase gene may be replaced with respective equivalent
codons. For example, the aconitase or acetaldehyde dehydrogenase
gene may be a gene modified so that it has optimal codons according
to codon frequencies in the chosen host.
The aforementioned descriptions concerning conservative variants of
the genes and proteins can be similarly applied to variants of
arbitrary proteins such as L-amino acid biosynthesis system enzymes
and ethanol metabolic enzymes and genes encoding them.
<1-3> Ethanol-Utilizing Ability
The bacterium has an ethanol-utilizing ability. The expression that
"a bacterium has an ethanol-utilizing ability" means that the
bacterium can grow in a minimal medium containing ethanol as the
sole carbon source. The bacterium may inherently have an
ethanol-utilizing ability, or it may have been modified so that it
has an ethanol-utilizing ability. A bacterium having an
ethanol-utilizing ability can be obtained by, for example,
imparting an ethanol-utilizing ability to any of such bacteria as
mentioned above, or enhancing an ethanol-utilizing ability of the
same.
An ethanol-utilizing ability can be imparted or enhanced by
modifying a bacterium so that the activity or activities of one or
more of the ethanol metabolic enzymes are increased. That is, the
bacterium may have been modified so that the activity or activities
of one or more ethanol metabolic enzymes are increased.
Examples of the ethanol metabolic enzymes include alcohol
dehydrogenase and CoA-dependent acetaldehyde dehydrogenase.
The term "alcohol dehydrogenase" refers to a protein having an
activity for reversibly catalyzing the reaction of generating
acetaldehyde from ethanol by using NAD.sup.+ or NADP.sup.+ as an
electron acceptor (EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.71, etc.). This
activity is also referred to as "alcohol dehydrogenase activity".
The alcohol dehydrogenase activity can be measured by, for example,
measuring ethanol-dependent reduction of NAD.sup.+ (Clark D, Cronan
J E Jr., J. Bacteriol., 1980, January; 141(1):177-83).
The term "CoA-dependent acetaldehyde dehydrogenase" refers to a
protein having an activity for reversibly catalyzing the reaction
of generating acetyl-CoA from acetaldehyde by using NAD.sup.+ or
NADP.sup.+ as an electron acceptor (EC 1.2.1.10). This activity is
also referred to as "CoA-dependent acetaldehyde dehydrogenase
activity". The CoA-dependent acetaldehyde dehydrogenase activity
can be measured by, for example, measuring acetaldehyde- and
CoA-dependent reduction of NAD.sup.+ (Rudolph F B, Purich D L,
Fromm H J., J. Biol. Chem., 1968, Nov. 10; 243 (21):5539-45).
Examples of the ethanol metabolic enzymes include AdhE protein,
which is encoded by adhE gene. The AdhE protein is a bi-functional
enzyme, and has both the alcohol dehydrogenase activity and
CoA-dependent acetaldehyde dehydrogenase activity. Examples of the
AdhE protein include, for example, AdhE proteins of bacteria
belonging to the family Enterobacteriaceae such as Escherichia
coli, Pantoea ananatis, Pectobacterium atrosepticum (formerly,
Envinia carotovora), and Salmonella enterica.
The adhE gene of the Escherichia coli K-12 MG1655 strain
corresponds to the complementary sequence of the sequence of the
positions 1295446 to 1298121 in the genome sequence registered at
the NCBI database as GenBank accession NC_000913 (VERSION
NC_000913.3 GI: 556503834). The AdhE protein of the MG1655 strain
is registered as GenBank accession NP_415757 (version NP_415757.1
GI: 16129202). The nucleotide sequence of the adhE gene and the
amino acid sequence of the AdhE protein of the MG1655 strain are
shown as SEQ ID NOS: 45 and 46, respectively.
The adhE gene of the Pantoea ananatis LMG 20103 strain corresponds
to the sequence of the positions 2335387 to 2338071 in the genome
sequence registered at the NCBI database as GenBank accession
NC_013956 (VERSION NC_013956.2 GI: 332139403). The AdhE protein of
the LMG 20103 strain is registered as GenBank accession
YP_003520384 (version YP_003520384.1 GI: 291617642). The nucleotide
sequence of the adhE gene and the amino acid sequence of the AdhE
protein of the LMG 20103 strain are shown as SEQ ID NOS: 47 and 48,
respectively.
The adhE gene of the Pectobacterium atrosepticum SCRI1043 strain
corresponds to the sequence of the positions 2634501 to 2637176 in
the genome sequence registered at the NCBI database as GenBank
accession NC_004547 (VERSION NC_004547.2 GI: 50119055). The AdhE
protein of the SCRI1043 strain is registered as GenBank accession
YP_050421 (version YP_050421.1 GI: 50121254). The nucleotide
sequence of the adhE gene and the amino acid sequence of the AdhE
protein of the SCRI1043 strain are shown as SEQ ID NOS: 49 and 50,
respectively.
An adhE gene homologue of the Salmonella enterica serovar Typhi
CT18 strain is registered as adh gene at a database. This adhE gene
homologue is regarded as the adhE gene. The adhE gene of the
Salmonella enterica serovar Typhi CT18 strain corresponds to the
complementary sequence of the sequence of the positions 1259893 to
1262571 in the genome sequence registered at the NCBI database as
GenBank accession NC_003198 (VERSION NC_003198.1 GI: 16762629). The
AdhE protein of the CT18 strain is registered as GenBank accession
NP_455751 (version NP_455751.1 GI: 16760134). The nucleotide
sequence of the adhE gene and the amino acid sequence of AdhE
protein of the CT18 strain are shown as SEQ ID NOS: 51 and 52,
respectively.
The result of alignment of these AdhE proteins is shown in FIG.
2A-2C. The homologies of the amino acid sequence of the AdhE
protein of the Escherichia coli K-12 MG1655 strain to the amino
acid sequences of the AdhE proteins of the AdhE proteins of the
Pantoea ananatis LMG 20103 strain, Pectobacterium atrosepticum
SCRI1043 strain, and Salmonella enterica serovar Typhi CT18 strain
are 89.0%, 89.1%, and 97.2%, respectively.
The ethanol metabolic enzyme may be a conservative variant of any
of the ethanol metabolic enzymes exemplified above such as the AdhE
proteins of bacteria belonging to the family Enterobacteriaceae
exemplified above. For example, the AdhE protein may be a protein
having the amino acid sequence shown as SEQ ID NO: 46, 48, 50, or
52, but including substitution, deletion, insertion, or addition of
one or several amino acid residues at one or several positions. For
the variants of genes and proteins, the descriptions concerning
conservative variants of aconitase and acetaldehyde dehydrogenase,
and genes encoding them mentioned above can be similarly
applied.
The bacterium has an ethanol-utilizing ability under aerobic
conditions, that is, it can aerobically utilize ethanol. The
expression that "a bacterium has an ethanol-utilizing ability under
aerobic conditions" means that the bacterium can grow in a minimal
medium containing ethanol as a sole carbon source under aerobic
conditions. The expression that "a bacterium has an
ethanol-utilizing ability under aerobic conditions" may mean that,
for example, the specific activity of alcohol dehydrogenase in a
cell-free extract prepared from cells of the bacterium obtained by
aerobic culture is, for example, 1.5 U/mg protein or higher, 5 U/mg
protein or higher, or 10 U/mg protein or higher. One unit of the
alcohol dehydrogenase activity is defined as generation of 1 nmol
of NADH in 1 minute under the aforementioned activity measurement
conditions (Clark D, Cronan J E Jr., J. Bacteriol., 1980, January;
141(1):177-83). The term "aerobic conditions" refers to culture
conditions that oxygen is supplied to the culture system by, for
example, aeration, shaking, and/or stirring. The bacterium may
inherently have an ethanol-utilizing ability under aerobic
conditions, or it may have been modified so that it has an
ethanol-utilizing ability under aerobic conditions. For example,
although Escherichia coli typically cannot aerobically utilize
ethanol, Escherichia coli may be modified so that it can
aerobically utilize ethanol.
An ethanol-utilizing ability under aerobic conditions can be
imparted or enhanced by modifying a bacterium so that the activity
or activities of one or more kinds of enzymes selected from ethanol
metabolic enzymes are increased under aerobic conditions. That is,
the bacterium may have been modified so that the activity or
activities of one or more ethanol metabolic enzymes are increased
under aerobic conditions.
An ethanol-utilizing ability under aerobic conditions can be
imparted or enhanced by, for example, modifying a bacterium so that
the bacterium has an adhE gene that is expressed under the control
of a promoter that functions under aerobic conditions.
Such modification can be attained by, for example, replacing the
native promoter of an adhE gene on a bacterial genome with a
promoter that functions under aerobic conditions. Alternatively, an
adhE gene ligated downstream from a promoter that functions under
aerobic conditions may be introduced into a bacterium, or an adhE
gene may be introduced downstream from a promoter that is present
on the bacterial genome and functions under the aerobic conditions.
As for replacement of a promoter or introduction of a gene, the
descriptions of "Methods for increasing activity of protein"
mentioned later can be referred to.
The promoter that functions under aerobic conditions is not
particularly limited, so long as it is able to express the adhE
gene under the aerobic conditions to such an extent that the
bacterium can utilize ethanol. Examples of the promoter that
functions under aerobic conditions include, for example, promoters
of genes of the glycolysis system, pentose phosphate pathway, TCA
cycle, and amino acid biosynthesis systems, and the P.sub.14
promoter (SEQ ID NO: 1) used in the Examples section. Examples of
the promoter that functions under aerobic conditions also include,
for example, such strong promoters as T7 promoter, trp promoter,
lac promoter, thr promoter, tac promoter, trc promoter, tet
promoter, araBAD promoter, rpoH promoter, PR promoter, and PL
promoter.
An ethanol-utilizing ability under aerobic conditions can be
imparted or enhanced by, for example, modifying a bacterium so as
to harbor an adhE gene encoding an AdhE protein having a mutation
for improving resistance to inactivation under aerobic conditions.
The "mutation for improving resistance to inactivation under
aerobic conditions" is also referred to as "aerobic resistance
mutation".
An AdhE protein having an aerobic resistance mutation is also
referred to as "mutant AdhE protein". A gene encoding a mutant AdhE
protein is also referred to as "mutant adhE gene."
An AdhE protein not having any aerobic resistance mutation is also
referred to as "wild-type AdhE protein". A gene encoding a
wild-type AdhE protein is also referred to as "wild-type adhE
gene". The term "wild-type" is used to distinguish from a "mutant"
gene or protein, and a "wild-type" gene or protein is not limited
to one obtained from the nature so long as it does not have any
aerobic resistance mutation. Examples of the wild-type AdhE protein
include, for example, AdhE proteins of the bacteria belonging to
the family Enterobacteriaceae exemplified above. Any of
conservative variants of AdhE proteins of the bacteria belonging to
the family Enterobacteriaceae exemplified above is regarded as a
wild-type AdhE protein, so long as it does not have any aerobic
resistance mutation.
Examples of the aerobic resistance mutation include a mutation
wherein an amino acid residue corresponding to the glutamic acid
residue at position 568 in the amino acid sequence of the wild-type
AdhE protein, such as SEQ ID NO: 46 of the AdhE protein of
Escherichia coli K-12 MG1655 strain, is replaced with an amino acid
residue other than glutamic acid and aspartic acid (WO2008/010565).
Examples of the amino acid residue at that position after the
replacement include K (Lys), R (Arg), H (His), A (Ala), V (Val), L
(Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), W
(Trp), Y (Tyr), C (Cys), M (Met), N (Asn), and Q (Gln). The amino
acid residue at that position after the replacement may be, for
example, lysine. When the amino acid residue at that position after
the replacement is lysine residue, the mutation is also referred to
as "Glu568Lys" or "E568K" mutation.
The mutant AdhE protein may further have an additional mutation
selected from the following mutations:
(A) a mutation that in the amino acid sequence of a wild-type AdhE
protein, such as the amino acid sequence of SEQ ID NO: 46, an amino
acid residue corresponding to the glutamic acid residue at position
560 is replaced with another amino acid residue,
(B) a mutation that in the amino acid sequence of a wild-type AdhE
protein, such as the amino acid sequence of SEQ ID NO: 46, an amino
acid residue corresponding to the phenylalanine residue at position
566 is replaced with another amino acid residue,
(C) a mutation that in the amino acid sequence of a wild-type AdhE
protein, such as the amino acid sequence of SEQ ID NO: 46, amino
acid residues corresponding to the glutamic acid residue at
position 22, methionine residue at position 236, tyrosine residue
at position 461, isoleucine residue at position 554, and alanine
residue at position 786 are replaced with respective other amino
acid residues;
(D) a combination of these mutations.
As for the aforementioned additional mutations, examples of the
amino acid residue after the replacement include K (Lys), R (Arg),
H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T
(Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D
(Asp), E (Glu), N (Asn), and Q (Gln), provided that the amino acid
residue after the replacement must differ from the amino acid
residue before the replacement. In the case of the aforementioned
mutation (A), the amino acid residue existing after the replacement
may be, for example, lysine residue. In the case of the
aforementioned mutation (B), the amino acid residue existing after
the replacement may be, for example, valine residue. In the case of
the aforementioned mutation (C), the amino acid residues existing
after the replacement may be, for example, glycine residue for
position 22 (Glu22Gly), valine residue for position 236
(Met236Val), cysteine residue for position 461 (Tyr461Cys), serine
residue for position 554 (Ile554Ser), and valine residue for
position 786 (Ala786Val).
In the amino acid sequence of an arbitrary wild-type AdhE protein,
the term "amino acid residue corresponding to the amino acid
residue at position n in the amino acid sequence of SEQ ID NO: 46"
refers to an amino acid residue corresponding to the amino acid
residue at position n in the amino acid sequence of SEQ ID NO: 46
determined in alignment of the amino acid sequence of the objective
wild-type AdhE protein and the amino acid sequence of SEQ ID NO:
46. That is, the positions of amino acid residues defined in the
aforementioned mutations do not necessarily represent the absolute
positions in the amino acid sequence of a wild-type AdhE protein,
but represent the relative positions determined on the basis of the
amino acid sequence of SEQ ID NO: 46. For example, when one amino
acid residue of the wild-type AdhE protein consisting of the amino
acid sequence of SEQ ID NO: 46 is deleted at a position on the
N-terminus side of position n, the amino acid residue originally at
position n becomes the (n-1)th amino acid residue counted from the
N-terminus, but it is still regarded as the "amino acid residue
corresponding to the amino acid residue at position n in the amino
acid sequence of SEQ ID NO: 46". Similarly, for example, when an
amino acid residue at position 567 in the amino acid sequence of an
AdhE protein homologue of a certain microorganism corresponds to
the amino acid residue at position 568 in the amino acid sequence
of SEQ ID NO: 46, that amino acid residue is regarded as the "amino
acid residue corresponding to the amino acid residue at position
568 in the amino acid sequence shown as SEQ ID NO: 46" in the AdhE
protein homologue.
Such alignment can be performed by, for example, using known gene
analysis software. Specific examples of such gene analysis software
include DNASIS produced by Hitachi Solutions, GENETYX produced by
Genetyx, ClustalW opened to the public by DDBJ, and so forth
(Elizabeth C. Tyler et al., Computers and Biomedical Research,
24(1), 72-96, 1991; Barton G J. et al., Journal of Molecular
Biology, 198(2), 327-37, 1987; Thompson J D et al., Nucleic Acid
Research, 22(22), 4673-80, 1994).
A mutant adhE gene can be obtained by, for example, modifying a
wild-type adhE gene so that the AdhE protein encoded by the
wild-type adhE gene has an aerobic resistance mutation. The
wild-type adhE gene to be modified can be obtained by, for example,
cloning from an organism having the wild-type adhE gene, or
chemical synthesis. A mutant adhE gene may also be directly
obtained by, for example, chemical synthesis, or the like.
Modification of a gene can be performed by a known method. For
example, by the site-specific mutagenesis method, an objective
mutation can be introduced into a target site of DNA. Examples of
the site-specific mutagenesis method include a method of using PCR
(Higuchi, R., 61, in PCR Technology, Erlich, H. A. Eds., Stockton
Press (1989); Carter P., Meth. in Enzymol., 154, 382 (1987)), and a
method of using a phage (Kramer, W. and Frits, H. J., Meth. in
Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol.,
154, 367 (1987)).
A mutant adhE gene is introduced into the bacterium in such a
manner that the gene can be expressed. Specifically, the gene can
be introduced into the bacterium so that it is expressed under the
control of a promoter that functions under aerobic conditions. As
for introduction of a gene, the descriptions of "Methods for
increasing activity of protein" mentioned later can be referred
to.
<1-4> Other Modifications
The bacterium may also have been modified so that the activity of
pyruvate synthase (also referred to as "PS") and/or
pyruvate:NADP.sup.+ oxidoreductase (also referred to as "PNO") is
increased (WO2009/031565).
The term "pyruvate synthase" refers to an enzyme reversibly
catalyzing the reaction of generating pyruvic acid from acetyl-CoA
and CO.sub.2 using the reduced ferredoxin or reduced flavodoxin as
an electron donor (EC 1.2.7.1). PS is also referred to as pyruvate
oxidoreductase, pyruvate ferredoxin oxidoreductase, or pyruvate
flavodoxin oxidoreductase. The activity of PS can be measured
according to, for example, the method of Yoon et al. (Yoon, K. S.
et al., 1997, Arch. Microbiol., 167:275-279).
Examples of a gene encoding PS (PS gene) include PS genes of
bacteria having the reductive TCA cycle such as Chlorobium tepidum
and Hydrogenobacter thermophilus, PS genes of bacteria belonging to
the family Enterobacteriaceae such as Escherichia coli, and PS
genes of autotrophic methanogens such as Methanococcus maripaludis,
Methanocaldococcus jannaschii, and Methanothermobacter
thermautotrophicus.
The term "pyruvate:NADP.sup.+ oxidoreductase" refers to an enzyme
reversibly catalyzing the reaction of generating pyruvic acid from
acetyl CoA and CO.sub.2 using NADPH or NADH as an electron donor
(EC 1.2.1.15). The pyruvate:NADP.sup.+ oxidoreductase is also
referred to as pyruvate dehydrogenase. The activity of PNO can be
measured by, for example, the method of Inui et al. (Inui, H., et
al., 1987, J. Biol. Chem., 262:9130-9135).
Examples of a gene encoding PNO (PNO gene) include the PNO gene of
Euglena gracilis, which is a photosynthetic eukaryotic
microorganism and is also classified into protozoans (Nakazawa, M.
et al., 2000, FEBS Lett., 479:155-156), the PNO gene of a protist,
Cryptosporidium parvum (Rotte, C. et al., 2001, Mol. Biol. Evol.,
18:710-720, GenBank Accession No. AB021127), and a PNO homologous
gene of Bacillariophyta, Tharassiosira pseudonana (Ctrnacta, V. et
al., 2006, J. Eukaryot. Microbiol., 53:225-231).
Enhancement of the PS activity can also be attained by, besides the
methods of increasing the activities of proteins such as described
later, improving supply of the electron donor required for the PS
activity. For example, the PS activity can be enhanced by enhancing
the activity of recycling ferredoxin or flavodoxin of the oxidized
form to that of the reduced form, enhancing the ability to
biosynthesize ferredoxin or flavodoxin, or combination of them
(WO2009/031565).
Examples of a protein having the activity of recycling ferredoxin
or flavodoxin of the oxidized form to that of the reduced form
include ferredoxin NADP.sup.+ reductase. The term "ferredoxin
NADP.sup.+ reductase" refers to an enzyme that reversibly catalyzes
the reaction of converting ferredoxin or flavodoxin of the oxidized
form to that of the reduced form using NADPH as the electron donor
(EC 1.18.1.2). Ferredoxin NADP.sup.+ reductase is also referred to
as flavodoxin NADP.sup.+ reductase. The activity of ferredoxin
NADP+ reductase can be measured by, for example, the method of
Blaschkowski et al. (Blaschkowski, H. P. et al., 1982, Eur. J.
Biochem., 123:563-569).
Examples of a gene encoding ferredoxin NADP.sup.+ reductase
(ferredoxin NADP.sup.+ reductase gene) include the fpr gene of
Escherichia coli, the ferredoxin NADP+ reductase gene of
Corynebacterium glutamicum, and the NADPH-putidaredoxin reductase
gene of Pseudomonas putida (Koga, H. et al., 1989, J. Biochem.
(Tokyo) 106:831-836).
The ability to biosynthesize ferredoxin or flavodoxin can be
enhanced by enhancing the expression of a gene encoding ferredoxin
(ferredoxin gene) or a gene encoding flavodoxin (flavodoxin gene).
The ferredoxin gene or flavodoxin gene is not particularly limited,
so long as it encodes ferredoxin or flavodoxin that can be utilized
by PS and the electron donor recycling system.
Examples of the ferredoxin gene include the fdx gene and yfhL gene
of Escherichia coli, the fer gene of Corynebacterium glutamicum,
and ferredoxin genes of bacteria having the reductive TCA cycle
such as Chlorobium tepidum and Hydrogenobacter thermophilus.
Examples of the flavodoxin gene include the fldA gene and fldB gene
of Escherichia coli, and flavodoxin genes of bacteria having the
reductive TCA cycle.
The bacterium may also have been modified so that the activity of
ribonuclease G is reduced (JP2012-100537A).
The bacterium may also have been modified so as to harbor a mutant
ribosome S1 protein (JP2013-074795A).
The bacterium may also have been modified so that the intracellular
concentration of hydrogen peroxide is reduced (JP2014-036576A).
The aforementioned genes such as the PS gene, PNO gene, ferredoxin
NADP+ reductase gene, ferredoxin gene, and flavodoxin gene are not
limited to genes having the aforementioned genetic information and
genes having a known nucleotide sequence, and may be a variant
thereof, so long as the functions of the encoded proteins are not
degraded. For example, the genes may be a gene encoding a protein
having an amino acid sequence of a known protein, but including
substitution, deletion, insertion, or addition of one or several
amino acid residues at one or several positions. For the variants
of genes and proteins, the descriptions concerning conservative
variants of aconitase and acetaldehyde dehydrogenase, and genes
encoding them mentioned above can be similarly applied.
<1-5> Methods for Increasing Activity of Protein
Hereafter, the methods for increasing the activity of a protein
such as aconitase and acetaldehyde dehydrogenase will be
explained.
The expression "the activity of a protein is increased" means that
the activity of the protein per cell is increased as compared with
that of a non-modified strain such as a wild-type strain or
parental strain. The state that "the activity of a protein is
increased" may also be expressed as "the activity of a protein is
enhanced". Specifically, the expression "the activity of a protein
is increased" means that the number of molecules of the protein per
cell is increased, and/or the function of each molecule of the
protein is increased as compared with those of a non-modified
strain. That is, the term "activity" in the expression "the
activity of a protein is increased" is not limited to the catalytic
activity of the protein, but may also mean the transcription amount
of a gene (i.e. the amount of mRNA) encoding the protein, or the
translation amount of the protein (i.e. the amount of the protein).
Furthermore, the state that "the activity of a protein is
increased" includes not only a state that the activity of an
objective protein is increased in a strain inherently having the
activity of the objective protein, but also a state that the
activity of an objective protein is imparted to a strain not
inherently having the activity of the objective protein.
Furthermore, so long as the activity of the protein is eventually
increased, the activity of an objective protein inherently
contained in a host may be attenuated and/or eliminated, and then
an appropriate type of the objective protein may be imparted to the
host.
The degree of the increase in the activity of a protein is not
particularly limited, so long as the activity of the protein is
increased as compared with a non-modified strain. The activity of
the protein may be increased, for example, 1.5 times or more, 2
times or more, or 3 times or more, as compared with that of a
non-modified strain. Furthermore, when the non-modified strain does
not have the activity of the objective protein, it is sufficient
that the protein is produced as a result of introduction of the
gene encoding the protein, and for example, the protein may be
produced to such an extent that the activity thereof can be
measured.
The modification for increasing the activity of a protein can be
attained by, for example, increasing the expression of a gene
encoding the protein. The expression "the expression of a gene is
increased" means that the expression amount of the gene per cell is
increased as compared with that of a non-modified strain such as a
wild-type strain or parental strain. Specifically, the expression
"the expression of a gene is increased" may mean that the
transcription amount of the gene (i.e. the amount of mRNA) is
increased, and/or the translation amount of the gene (i.e. the
amount of the protein expressed from the gene) is increased. The
state that "the expression of a gene is increased" may also be
referred to as "the expression of a gene is enhanced". The
expression of a gene may be increased, for example, 1.5 times or
more, 2 times or more, or 3 times or more, as compared with that of
a non-modified strain. Furthermore, the phrase that "the expression
of a gene is increased" includes not only when the expression
amount of an objective gene is increased in a strain that
inherently expresses the objective gene, but also when the gene is
introduced into a strain that does not inherently express the
objective gene, and is then expressed. That is, the phrase "the
expression of a gene is increased" may also mean, for example, that
an objective gene is introduced into a strain that does not possess
the gene, and is then expressed.
The expression of a gene can be increased by, for example,
increasing the copy number of the gene.
The copy number of a gene can be increased by introducing the gene
into the chromosome of a host. A gene can be introduced into a
chromosome by, for example, using homologous recombination (Miller,
J. H., Experiments in Molecular Genetics, 1972, Cold Spring Harbor
Laboratory). Examples of the gene transfer method utilizing
homologous recombination include, for example, a method using a
linear DNA such as Red-driven integration (Datsenko, K. A., and
Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), a
method of using a plasmid containing a temperature sensitive
replication origin, a method of using a plasmid capable of
conjugative transfer, a method of using a suicide vector not having
a replication origin that functions in a host, and a transduction
method using a phage. Only one copy, or two or more copies of a
gene may be introduced. For example, by performing homologous
recombination using a sequence which is present in multiple copies
on a chromosome as a target, multiple copies of a gene can be
introduced into the chromosome. Examples of such a sequence which
is present in multiple copies on a chromosome include repetitive
DNAs, and inverted repeats located at the both ends of a
transposon. Alternatively, homologous recombination may be
performed by using an appropriate sequence on a chromosome such as
a gene unnecessary for production of an objective substance as a
target. Furthermore, a gene can also be randomly introduced into a
chromosome by using a transposon or Mini-Mu (Japanese Patent
Laid-open (Kokai) No. 2-109985, U.S. Pat. No. 5,882,888, EP 805867
B1).
Introduction of a target gene into a chromosome can be confirmed by
Southern hybridization using a probe having a sequence
complementary to the whole gene or a part thereof, PCR using
primers prepared on the basis of the sequence of the gene, or the
like.
Furthermore, the copy number of a gene can also be increased by
introducing a vector containing the gene into a host. For example,
the copy number of a target gene can be increased by ligating a DNA
fragment containing the target gene with a vector that functions in
a host to construct an expression vector of the gene, and
transforming the host with the expression vector. The DNA fragment
containing the target gene can be obtained by, for example, PCR
using the genomic DNA of a microorganism having the target gene as
the template. As the vector, a vector autonomously replicable in
the cell of the host can be used. The vector is preferably a
multi-copy vector. Furthermore, the vector preferably has a marker
such as an antibiotic resistance gene for selection of
transformant. Furthermore, the vector may have a promoter and/or
terminator for expressing the introduced gene. The vector may be,
for example, a vector derived from a bacterial plasmid, a vector
derived from a yeast plasmid, a vector derived from a
bacteriophage, cosmid, phagemid, or the like. Specific examples of
vector autonomously replicable in Enterobacteriaceae bacteria such
as Escherichia coli include, for example, pUC19, pUC18, pHSG299,
pHSG399, pHSG398, pBR322, pSTV29 (all of these are available from
Takara Bio), pACYC184, pMW219 (NIPPON GENE), pTrc99A (Pharmacia),
pPROK series vectors (Clontech), pKK233-2 (Clontech), pET series
vectors (Novagen), pQE series vectors (QIAGEN), pACYC series
vectors, and the broad host spectrum vector RSF1010.
When a gene is introduced, it is sufficient that the gene is
expressibly harbored by the bacterium. Specifically, it is
sufficient that the gene is introduced so that it is expressed
under control by a promoter sequence that functions in the chosen
bacterium. The promoter may be a promoter derived from the host, or
a heterogenous promoter. The promoter may be the native promoter of
the gene to be introduced, or a promoter of another gene. Examples
of the promoter include promoters of genes of the glycolysis
system, pentose phosphate pathway, TCA cycle, and amino acid
biosynthesis systems, and the P.sub.14 promoter (SEQ ID NO: 1) used
in the Examples section. As the promoter, for example, such a
stronger promoter as mentioned later may also be used.
A terminator for termination of gene transcription may be located
downstream of the gene. The terminator is not particularly limited
so long as it functions in the bacterium of the present invention.
The terminator may be a terminator derived from the host, or a
heterogenous terminator. The terminator may be the native
terminator of the gene to be introduced, or a terminator of another
gene. Specific examples of the terminator include, for example, T7
terminator, T4 terminator, fd phage terminator, tet terminator, and
trpA terminator.
Vectors, promoters, and terminators available in various
microorganisms are disclosed in detail in "Fundamental Microbiology
Vol. 8, Genetic Engineering, KYORITSU SHUPPAN CO., LTD, 1987", and
those can be used.
Furthermore, when two or more of genes are introduced, it is
sufficient that the genes each are expressibly harbored by the
bacterium. For example, all the genes may be carried by a single
expression vector or a chromosome. Furthermore, the genes may be
separately carried by two or more expression vectors, or separately
carried by a single or two or more expression vectors and a
chromosome. An operon constituted by two or more genes may also be
introduced. The case of "introducing two or more genes" include,
for example, cases of introducing respective genes encoding two or
more kinds of enzymes, introducing respective genes encoding two or
more subunits constituting a single enzyme, and a combination of
the foregoing cases.
The gene to be introduced is not particularly limited so long as it
encodes a protein that functions in the host. The gene to be
introduced may be a gene derived from the host, or may be a
heterogenous gene. The gene to be introduced can be obtained by,
for example, PCR using primers designed on the basis of the
nucleotide sequence of the gene, and using the genomic DNA of an
organism having the gene, a plasmid carrying the gene, or the like
as a template. The gene to be introduced may also be totally
synthesized, for example, on the basis of the nucleotide sequence
of the gene (Gene, 60(1), 115-127 (1987)). The obtained gene can be
used as it is, or after being modified as required.
In addition, when a protein functions as a complex consisting of a
plurality of subunits, a part or all of the plurality of subunits
may be modified, so long as the activity of the protein is
eventually increased. That is, for example, when the activity of a
protein is increased by increasing the expression of a gene, the
expression of a part or all of the plurality of genes that encode
the subunits may be enhanced. It is usually preferable to enhance
the expression of all of the plurality of genes encoding the
subunits. Furthermore, the subunits constituting the complex may be
derived from a single kind of organism or two or more kinds of
organisms, so long as the complex has a function of the objective
protein. That is, for example, genes of the same organism encoding
a plurality of subunits may be introduced into a host, or genes of
different organisms encoding a plurality of subunits may be
introduced into a host.
Furthermore, the expression of a gene can be increased by improving
the transcription efficiency of the gene. In addition, the
expression of a gene can also be increased by improving the
translation efficiency of the gene. The transcription efficiency of
the gene and the translation efficiency of the gene can be improved
by, for example, modifying an expression control sequence of the
gene. The term "expression control sequence" collectively refers to
sites that affect the expression of a gene. Examples of the
expression control sequence include, for example, promoter,
Shine-Dalgarno (SD) sequence (also referred to as ribosome binding
site (RBS)), and spacer region between RBS and the start codon.
Expression control sequences can be identified by using a promoter
search vector or gene analysis software such as GENETYX. These
expression control sequences can be modified by, for example, a
method of using a temperature sensitive vector, or the Red driven
integration method (WO2005/010175).
The transcription efficiency of a gene can be improved by, for
example, replacing the promoter of the gene on a chromosome with a
stronger promoter. The term "stronger promoter" means a promoter
providing an improved transcription of a gene compared with an
inherently existing wild-type promoter of the gene. Examples of
stronger promoters include, for example, the known high expression
promoters such as T7 promoter, trp promoter, lac promoter, thr
promoter, tac promoter, trc promoter, tet promoter, araBAD
promoter, rpoH promoter, PR promoter, and PL promoter. Furthermore,
as the stronger promoter, a highly-active type of an existing
promoter may also be obtained by using various reporter genes. For
example, by making the -35 and -10 regions in a promoter region
closer to the consensus sequence, the activity of the promoter can
be enhanced (WO00/18935). Examples of highly active-type promoter
include various tac-like promoters (Katashkina J I et al., Russian
Federation Patent Application No. 2006134574) and pnlp8 promoter
(WO2010/027045). Methods for evaluating the strength of promoters
and examples of strong promoters are described in the paper of
Goldstein et al. (Prokaryotic Promoters in Biotechnology,
Biotechnol. Annu. Rev., 1, 105-128 (1995)), and so forth.
The translation efficiency of a gene can be improved by, for
example, replacing the Shine-Dalgarno (SD) sequence (also referred
to as ribosome binding site (RBS)) for the gene on a chromosome
with a stronger SD sequence. The "stronger SD sequence" means a SD
sequence that provides an improved translation of mRNA compared
with the inherently existing wild-type SD sequence of the gene.
Examples of stronger SD sequences include, for example, RBS of the
gene 10 derived from phage T7 (Olins P. O. et al, Gene, 1988, 73,
227-235). Furthermore, it is known that substitution, insertion, or
deletion of several nucleotides in a spacer region between RBS and
the start codon, especially in a sequence immediately upstream of
the start codon (5'-UTR), significantly affects the stability and
translation efficiency of mRNA, and hence, the translation
efficiency of a gene can also be improved by modifying them.
The translation efficiency of a gene can also be improved by, for
example, modifying codons. In Escherichia coli etc., a clear codon
bias exists among the 61 amino acid codons found within the
population of mRNA molecules, and the level of cognate tRNA appears
directly proportional to the frequency of codon usage (Kane, J. F.,
Curr. Opin. Biotechnol., 6 (5), 494-500 (1995)). That is, if there
is a large amount of mRNA containing an excess amount of rare
codons, a translational problem may arise. According to the recent
research, it is suggested that clusters of AGG/AGA, CUA, AUA, CGA,
or CCC codons may especially reduce both the quantity and quality
of a synthesized protein. Such a problem occurs especially at the
time of expression of a heterologous gene. Therefore, in the case
of heterogenous expression of a gene or the like, the translation
efficiency of the gene can be improved by replacing a rare codon
present in the gene with a synonymous codon more frequently used.
That is, the gene to be introduced may be modified, for example, so
as to contain optimal codons according to the frequencies of codons
observed in a host to be used. Codons can be replaced by, for
example, the site-specific mutation method for introducing an
objective mutation into an objective site of DNA. Examples of the
site-specific mutation method include the method utilizing PCR
(Higuchi, R., 61, in PCR Technology, Erlich, H. A. Eds., Stockton
Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)), and
the method utilizing phage (Kramer, W. and Frits, H. J., Meth. in
Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol.,
154, 367 (1987)). Alternatively, a gene fragment in which objective
codons are replaced may be totally synthesized. Frequencies of
codons in various organisms are disclosed in the "Codon Usage
Database" (kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids
Res., 28, 292 (2000)).
Furthermore, the expression of a gene can also be increased by
amplifying a regulator that increases the expression of the gene,
or deleting or attenuating a regulator that reduces the expression
of the gene.
Such methods for increasing the gene expression as mentioned above
may be used independently or in an arbitrary combination.
Furthermore, the modification that increases the activity of a
protein can also be attained by, for example, enhancing the
specific activity of the enzyme. Enhancement of the specific
activity also includes desensitization to feedback inhibition. That
is, when a protein is subject to feedback inhibition by a
metabolite, the activity of the protein can be increased by making
the bacterium harbor a gene encoding a mutant protein that has been
desensitized to the feedback inhibition. The phrase
"desensitization to feedback inhibition" includes attenuation and
elimination of the feedback inhibition. A protein showing an
enhanced specific activity can be obtained by, for example,
searching various organisms. Furthermore, a highly-active type of
an existing protein may also be obtained by introducing a mutation
into the existing protein. The mutation to be introduced may be,
for example, substitution, deletion, insertion, or addition of one
or several amino acid residues at one or several position of the
protein. The mutation can be introduced by, for example, such a
site-specific mutation method as mentioned above. The mutation may
also be introduced by, for example, a mutagenesis treatment.
Examples of the mutagenesis treatment include irradiation of X-ray,
irradiation of ultraviolet, and a treatment with a mutation agent
such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl
methanesulfonate (EMS), and methyl methanesulfonate (MMS).
Furthermore, a random mutation may be induced by directly treating
DNA in vitro with hydroxylamine. Enhancement of the specific
activity may be independently used, or may be used in an arbitrary
combination with such methods for enhancing gene expression as
mentioned above.
The method for the transformation is not particularly limited, and
conventionally known methods can be used. There can be used, for
example, a method of treating recipient cells with calcium chloride
so as to increase the permeability thereof for DNA, which has been
reported for the Escherichia coli K-12 strain (Mandel, M. and Higa,
A., J. Mol. Biol., 1970, 53, 159-162), and a method of preparing
competent cells from cells which are in the growth phase, followed
by transformation with DNA, which has been reported for Bacillus
subtilis (Duncan, C. H., Wilson, G A. and Young, F. E., Gene, 1977,
1:153-167). Alternatively, there can also be used a method of
making DNA-recipient cells into protoplasts or spheroplasts, which
can easily take up recombinant DNA, followed by introducing a
recombinant DNA into the DNA-recipient cells, which is known to be
applicable to Bacillus subtilis, actinomycetes, and yeasts (Chang,
S. and Choen, S. N., 1979, Mol. Gen. Genet., 168:111-115; Bibb, M.
J., Ward, J. M. and Hopwood, O. A., 1978, Nature, 274:398-400;
Hinnen, A., Hicks, J. B. and Fink, G R., 1978, Proc. Natl. Acad.
Sci. USA, 75:1929-1933). Furthermore, the electric pulse method
reported for coryneform bacteria (Japanese Patent Laid-open (Kokai)
No. 2-207791) can also be used.
An increase in the activity of a protein can be confirmed by
measuring the activity of the protein.
An increase in the activity of a protein can also be confirmed by
confirming an increase in the expression of a gene encoding the
protein. An increase in the expression of a gene can be confirmed
by confirming an increase in the transcription amount of the gene,
or by confirming an increase in the amount of a protein expressed
from the gene.
An increase of the transcription amount of a gene can be confirmed
by comparing the amount of mRNA transcribed from the gene with that
of a non-modified strain such as a wild-type strain or parental
strain. Examples of the method for evaluating the amount of mRNA
include Northern hybridization, RT-PCR, and so forth (Sambrook, J.,
et al., Molecular Cloning A Laboratory Manual/Third Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001).
The amount of mRNA may increase, for example, 1.5 times or more, 2
times or more, or 3 times or more, as compared with that of a
non-modified strain.
An increase in the amount of a protein can be confirmed by Western
blotting using antibodies (Molecular Cloning, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of
the protein may increase, for example, 1.5 times or more, 2 times
or more, or 3 times or more, as compared with that of a
non-modified strain.
The aforementioned methods for increasing the activity of a protein
can be used for enhancement of the activities of arbitrary proteins
such as L-amino acid biosynthesis enzymes, and enhancement of the
expression of arbitrary genes such as genes encoding those
arbitrary proteins, besides enhancement of the aconitase and
acetaldehyde dehydrogenase activities.
<1-4> Method for Reducing Activity of Protein
Hereafter, the methods for reducing the activity of a protein will
be explained.
The expression "the activity of a protein is reduced" means that
the activity of the protein per cell is reduced as compared with
that of a non-modified strain such as a wild-type strain and
parental strain. The state that "the activity of a protein is
reduced" also includes a state that the activity of the protein has
completely disappeared. Specifically, the expression "the activity
of a protein is reduced" means that the number of molecules of the
protein per cell is reduced, and/or the function of each molecule
of the protein is reduced as compared with those of a non-modified
strain. That is, the term "activity" in the expression "the
activity of a protein is reduced" is not limited to the catalytic
activity of the protein, but may also mean the transcription amount
of a gene (i.e. the amount of mRNA) encoding the protein or the
translation amount of the protein (i.e. the amount of the protein).
The state that "the number of molecules of the protein per cell is
reduced" also includes a state that the protein does not exist at
all. The state that "the function of each molecule of the protein
is reduced" also includes a state that the function of each protein
molecule has completely disappeared. The degree of the reduction in
the activity of a protein is not particularly limited, so long as
the activity is reduced as compared with that of a non-modified
strain. The activity of a protein may be reduced to, for example,
50% or less, 20% or less, 10% or less, 5% or less, or 0%, as
compared with that of a non-modified strain.
The modification for reducing the activity of a protein can be
attained by, for example, reducing the expression of a gene
encoding the protein. The expression "the expression of a gene is
reduced" means that the expression of the gene per cell is reduced
as compared with that of a non-modified strain such as a wild-type
strain and parental strain. The expression "the expression of a
gene is reduced" may specifically mean that the transcription
amount of the gene (i.e. the amount of mRNA) is reduced, and/or the
translation amount of the gene (i.e. the amount of the protein
expressed from the gene) is reduced. The state that "the expression
of a gene is reduced" also includes a state that the gene is not
expressed at all. The state that "the expression of a gene is
reduced" is also referred to as "the expression of a gene is
attenuated". The expression of a gene may be reduced to, for
example, 50% or less, 20% or less, 10% or less, 5% or less, or 0%,
as compared with that of a non-modified strain.
The reduction in gene expression may be due to, for example, a
reduction in the transcription efficiency, a reduction in the
translation efficiency, or a combination of these. The expression
of a gene can be reduced by modifying an expression control
sequence of the gene such as promoter, Shine-Dalgarno (SD) sequence
(also referred to as ribosome-binding site (RBS)), and spacer
region between RBS and the start codon of the gene. When an
expression control sequence is modified, one or more nucleotides,
two or more nucleotides, three or more nucleotides, of the
expression control sequence are modified. Furthermore, a part of or
the entire expression control sequence may be deleted. The
expression of a gene can also be reduced by, for example,
manipulating a factor responsible for expression control. Examples
of the factor responsible for expression control include low
molecules responsible for transcription or translation control
(inducers, inhibitors, etc.), proteins responsible for
transcription or translation control (transcription factors etc.),
nucleic acids responsible for transcription or translation control
(siRNA etc.), and so forth. Furthermore, the expression of a gene
can also be reduced by, for example, introducing a mutation that
reduces the expression of the gene into the coding region of the
gene. For example, the expression of a gene can be reduced by
replacing a codon in the coding region of the gene with a
synonymous codon used less frequently in a host. Furthermore, for
example, the gene expression may be reduced due to disruption of a
gene as described later.
The modification for reducing the activity of a protein can also be
attained by, for example, disrupting a gene encoding the protein.
The expression "a gene is disrupted" means that a gene is modified
so that a protein that can normally function is not produced. The
state that "a protein that normally functions is not produced"
includes a state that the protein is not produced at all from the
gene, and a state that the protein of which the function (such as
activity or property) per molecule is reduced or eliminated is
produced from the gene.
Disruption of a gene can be attained by, for example, deleting a
part of or the entire coding region of the gene on a chromosome.
Furthermore, the entire gene including sequences upstream and
downstream from the gene on a chromosome may be deleted. The region
to be deleted may be any region such as an N-terminus region, an
internal region, or a C-terminus region, so long as the activity of
the protein can be reduced. Deletion of a longer region can usually
more surely inactivate the gene. Furthermore, reading frames of the
sequences upstream and downstream from the region to be deleted may
not be the same.
Disruption of a gene can also be attained by, for example,
introducing a mutation for an amino acid substitution (missense
mutation), a stop codon (nonsense mutation), a frame shift mutation
which adds or deletes one or two nucleotide residues, or the like
into the coding region of the gene on a chromosome (Journal of
Biological Chemistry, 272:8611-8617 (1997); Proceedings of the
National Academy of Sciences, USA, 95 5511-5515 (1998); Journal of
Biological Chemistry, 26 116, 20833-20839 (1991)).
Disruption of a gene can also be attained by, for example,
inserting another sequence into a coding region of the gene on a
chromosome. Site of the insertion may be in any region of the gene,
and insertion of a longer region can usually more surely inactivate
the gene. Reading frames of the sequences upstream and downstream
from the insertion site may not be the same. The other sequence is
not particularly limited so long as the chosen sequence reduces or
eliminates the activity of the encoded protein, and examples
thereof include, for example, a marker gene such as antibiotic
resistance genes, and a gene useful for production of an objective
sub stance.
Such modification of a gene on a chromosome as described above can
be attained by, for example, preparing a deficient type gene
modified so that it is unable to produce a protein that normally
functions, and transforming a host with a recombinant DNA
containing the deficient type gene to cause homologous
recombination between the deficient type gene and the wild-type
gene on a chromosome and thereby substitute the deficient type gene
for the wild-type gene on the chromosome. In this procedure, if a
marker gene selected according to the characteristics of the host
such as auxotrophy is included in the recombinant DNA, the
operation becomes easier. Examples of the deficient type gene
include a gene including deletion of all or a part of the gene,
gene including a missense mutation, gene including insertion of a
transposon or marker gene, gene including a nonsense mutation, and
gene including a frame shift mutation. The protein encoded by the
deficient type gene has a conformation different from that of the
wild-type protein, even if it is produced, and thus the function
thereof is reduced or eliminated. Such gene disruption based on
gene substitution utilizing homologous recombination has already
been established, and there are methods of using a linear DNA such
as a method called "Red driven integration" (Datsenko, K. A, and
Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)),
and a method utilizing the Red driven integration in combination
with an excision system derived from .lamda., phage (Cho, E. H.,
Gumport, R. I., Gardner, J. F., J. Bacteriol., 184:5200-5203
(2002)) (refer to WO2005/010175), a method of using a plasmid
having a temperature sensitive replication origin, a method of
using a plasmid capable of conjugative transfer, a method of
utilizing a suicide vector not having a replication origin that
functions in a host (U.S. Pat. No. 6,303,383, Japanese Patent
Laid-open (Kokai) No. 05-007491), and so forth.
Modification for reducing activity of a protein can also be
attained by, for example, a mutagenesis treatment. Examples of the
mutagenesis treatment include irradiation of X-ray or ultraviolet
and treatment with a mutation agent such as
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate
(EMS), and methyl methanesulfonate (MMS).
When a protein functions as a complex consisting of a plurality of
subunits, a part or all of the plurality of subunits may be
modified, so long as the activity of the protein is eventually
reduced. That is, for example, a part or all of a plurality of
genes that encode the respective subunits may be disrupted or the
like. Furthermore, when there is a plurality of isozymes of a
protein, a part or all of the activities of the plurality of
isozymes may be reduced, so long as the activity of the protein is
eventually reduced. That is, for example, a part or all of a
plurality of genes that encode the respective isozymes may be
disrupted or the like.
A reduction in the activity of a protein can be confirmed by
measuring the activity of the protein.
A reduction in the activity of a protein can also be confirmed by
confirming a reduction in the expression of a gene encoding the
protein. A reduction in the expression of a gene can be confirmed
by confirming a reduction in the transcription amount of the gene
or a reduction in the amount of the protein expressed from the
gene.
A reduction in the transcription amount of a gene can be confirmed
by comparing the amount of mRNA transcribed from the gene with that
of a non-modified strain. Examples of the method for evaluating the
amount of mRNA include Northern hybridization, RT-PCR, and so forth
(Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor (USA), 2001). The amount of mRNA can be reduced to,
for example, 50% or less, 20% or less, 10% or less, 5% or less, or
0%, as compared with that of a non-modified strain.
A reduction in the amount of a protein can be confirmed by Western
blotting using antibodies (Molecular Cloning, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor (USA) 2001). The amount of the
protein can be reduced to, for example, 50% or less, 20% or less,
10% or less, 5% or less, or 0%, as compared with that of a
non-modified strain.
Disruption of a gene can be confirmed by determining nucleotide
sequence of a part or the whole of the gene, restriction enzyme
map, full length, or the like of the gene depending on the means
used for the disruption.
The aforementioned methods for reducing the activity of a protein
as mentioned above can be applied to reduction in the activities of
arbitrary proteins such as an enzyme that catalyzes a reaction
branching away from the biosynthesis pathway of an objective
L-amino acid to generate a compound other than the objective
L-amino acid, and reduction in the expression of arbitrary genes
such as genes encoding those arbitrary proteins.
<2> Method for Producing L-Amino Acid of the Present
Invention
The method of the present invention is a method for producing an
L-amino acid by culturing the bacterium in a medium containing
ethanol to generate and accumulate the L-amino acid in the medium
or cells of the bacterium, and collecting the L-amino acid from the
medium or the cells. One kind of L-amino acid may be produced, or
two or more kinds of L-amino acids may be produced.
The medium is not particularly limited, so long as it contains
ethanol, the bacterium can proliferate in it, and an L-amino acid
can be produced. As the medium, for example, a usual medium used
for culture of bacteria and so forth can be used. The medium may
contain, in addition to ethanol, carbon source, nitrogen source,
phosphate source, and sulfur source, as well as components selected
from other various organic components and inorganic components as
required. Types and concentrations of the medium components may be
appropriately determined according to various conditions such as
type of the chosen bacterium and type of the L-amino acid to be
produced.
In the method, ethanol may be or may not be used as a sole carbon
source. That is, in the method, in addition to ethanol, another
carbon source may be used together. The other carbon source is not
particularly limited, so long as the bacterium can utilize, and an
L-amino acid can be produced. Specific examples of the other carbon
source include, for example, saccharides such as glucose, fructose,
sucrose, lactose, galactose, arabinose, blackstrap molasses,
hydrolysate of starch, and hydrolysate of biomass, organic acids
such as acetic acid, fumaric acid, citric acid, succinic acid, and
malic acid, alcohols such as glycerol and crude glycerol, and
aliphatic acids. When another carbon source is used, the ratio of
ethanol in the total carbon source may be, for example, 5% by
weight or more, 10% by weight or more, or 20% by weight or more,
30% by weight or more, 50% by weight or more. As the other carbon
source, one kind of carbon source may be used, or two or more kinds
of carbon sources may be used in combination.
The concentration of the carbon source in the medium is not
particularly limited, so long the bacterium can proliferate in the
medium, and an L-amino acid can be produced. The concentration of
the carbon source in the medium can be as high as possible in such
a range that the production of the L-amino acid is not inhibited.
The initial concentration of the carbon source in the medium may
be, for example, usually 1 to 30% (W/V), or 3 to 10% (W/V). Along
with the consumption of the carbon source that occurs as
fermentation advances, the carbon source may be continued to be
added.
Specific examples of the nitrogen source include, for example,
ammonium salts such as ammonium sulfate, ammonium chloride, and
ammonium phosphate, organic nitrogen sources such as peptone, yeast
extract, meat extract, and soybean protein decomposition products,
ammonia, and urea. Ammonia gas or aqueous ammonia used for
adjusting pH may also be used as the nitrogen source. As the
nitrogen source, a single kind of nitrogen source may be used, or
two or more kinds of nitrogen sources may be used in
combination.
Specific examples of the phosphate source include, for example,
phosphoric acid salts such as potassium dihydrogenphosphate and
dipotassium hydrogenphosphate, and phosphoric acid polymers such as
pyrophosphoric acid. As the phosphate source, a single kind of
phosphate source may be used, or two or more kinds of phosphate
sources may be used in combination.
Specific examples of the sulfur source include, for example,
inorganic sulfur compounds such as sulfates, thiosulfates, and
sulfites, and sulfur-containing amino acids such as cysteine,
cystine, and glutathione. As the sulfur source, a single kind of
sulfur source may be used, or two or more kinds of sulfur sources
may be used in combination.
Specific examples of other various organic components and inorganic
components include, for example, inorganic salts such as sodium
chloride and potassium chloride; trace metals such as iron,
manganese, magnesium, and calcium; vitamins such as vitamin B1,
vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin
B12; amino acids; nucleic acids; and organic components containing
those such as peptone, casamino acid, yeast extract, and soybean
protein decomposition product. As other various organic components
and inorganic components, a single kind of component may be used,
or two or more kinds of components may be used in combination.
Furthermore, when an auxotrophic mutant that requires an amino acid
or the like for growth thereof is used, a required nutrient can be
supplemented to the medium. For example, in many of
L-lysine-producing bacteria, the L-lysine biosynthetic pathway is
enhanced and the L-lysine degrading ability is attenuated.
Therefore, when such an L-lysine-producing bacterium is cultured,
for example, one or more amino acids such as L-threonine,
L-homoserine, L-isoleucine, and L-methionine can be added to the
medium.
Culture conditions are not particularly limited, so long as the
bacterium can proliferate, and an L-amino acid can be produced. The
culture can be performed with, for example, usual conditions used
for bacteria such as Escherichia coli. The culture conditions may
be appropriately determined depending on various conditions such as
the type of chosen bacterium and type of L-amino acid to be
produced.
The culture can be performed by using a liquid medium. At the time
of the culture, the bacterium cultured on a solid medium such as
agar medium may be directly inoculated into a liquid medium, or the
bacterium cultured in a liquid medium as seed culture may be
inoculated into a liquid medium for main culture. That is, the
culture may be performed as separate seed culture and main culture.
In such a case, the culture conditions of the seed culture and the
main culture may be or may not be the same. Amount of the bacterium
present in the medium at the time of the start of the culture is
not particularly limited. For example, a seed culture broth showing
an OD660 of 4 to 8 may be added to a medium for main culture at a
ratio of 0.1 to 30 mass %, or 1 to 10 mass %, at the time of the
start of the culture.
The culture can be performed as batch culture, fed-batch culture,
continuous culture, or a combination of these. The medium used at
the time of the start of the culture is also referred to as
"starting medium". The medium supplied to a culture system
(fermentation tank) in fed-batch culture or continuous culture is
also referred to as "feed medium". Furthermore, to supply a medium
to a culture system in fed-batch culture or continuous culture is
also referred to as to "feed". Furthermore, when the culture is
performed as separate seed culture and main culture, for example,
both the seed culture and the main culture may be performed as
batch culture. Alternatively, for example, the seed culture may be
performed as batch culture, and the main culture may be performed
as fed-batch culture or continuous culture.
The medium components each may be contained in the starting medium,
feed medium, or the both. The types of the components contained in
the starting medium may be or may not be the same as the types of
the components contained in the feed medium. The concentration of
each component contained in the starting medium may be or may not
be the same as the concentration of the component contained in the
feed medium. Furthermore, two or more kinds of feed media
containing different types and/or different concentrations of
components may be used. For example, when medium is intermittently
fed a plurality of times, the types and/or concentrations of
components contained in the feed media may be or may not be the
same.
The concentration of ethanol in the medium is not particularly
limited, so long as the bacterium can use ethanol as the carbon
source. Ethanol may be contained in the medium at a concentration
of, for example, 10 w/v % or lower, 5 w/v % or lower, or 2 w/v % or
lower. Also, ethanol may be contained in the medium at a
concentration of, for example, 0.2 w/v % or higher, 0.5 w/v % or
higher, or 1.0 w/v % or higher. Ethanol may be contained in the
starting medium, feed medium, or the both at a concentration within
the range exemplified above.
When ethanol is contained in the feed medium, ethanol may be
contained in the feed medium at such a concentration that, for
example, the ethanol concentration in the medium after feeding is 5
w/v % or lower, 2 w/v % or lower, or 1 w/v % or lower. When ethanol
is contained in the feed medium, ethanol may be contained in the
feed medium at such a concentration that, for example, the ethanol
concentration in the medium after feeding is 0.01 w/v % or higher,
0.02 w/v % or higher, or 0.05 w/v % or higher.
When ethanol is used as the sole carbon source, the ethanol
concentration may be within the range exemplified above. When
ethanol is used in combination with another carbon source, the
ethanol concentration may also be within the range exemplified
above. When ethanol is used in combination with another carbon
source, the ethanol concentration may also be within a range
defined by appropriately modifying the range exemplified above on
the basis of, for example, ratio of ethanol in the total carbon
source, or the like.
The ethanol concentration may be or may not be within a certain
range over the whole period of culture. For example, ethanol may
run short during a partial period of culture. The term "run short"
means that the amount of ethanol is smaller than the required
amount, and it may mean that, for example, the concentration in the
medium becomes zero. The term "partial period of culture" may refer
to, for example, 1% or less, 5% or less, 10% or less, 20% or less,
30% or less, or 50% or less of the whole period of the culture.
When the culture is performed as separate seed culture and main
culture, the term "whole period of the culture" may mean the whole
period of the main culture. It is preferred that, during a period
when ethanol runs short, another carbon source is present in a
sufficient amount. Even if ethanol runs short during a partial
period of culture as described above, culture performed under such
a condition falls within the scope of the expression "culture of a
bacterium in a medium containing ethanol", so long as there is a
culture period where the culture is performed in a medium
containing ethanol.
Concentration of various components such as ethanol can be measured
by gas chromatography (Hashimoto, K. et al., Biosci. Biotechnol.
Biochem., 1996, 70:22-30) or HPLC (Lin, J. T. et al., J.
Chromatogr. A., 1998, 808:43-49).
The culture can be, for example, aerobically performed. For
example, the culture can be performed as aeration culture or
shaking culture. The oxygen concentration can be controlled to be,
for example, 5 to 50%, or about 10%, of the saturated oxygen
concentration. pH of the medium may be, for example, 3 to 10, or
4.0 to 9.5. During the culture, pH of the medium can be adjusted as
required. pH of the medium can be adjusted by using various
alkaline and acidic substances such as ammonia gas, aqueous
ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate,
potassium bicarbonate, magnesium carbonate, sodium hydroxide,
calcium hydroxide, and magnesium hydroxide. The culture temperature
may be, for example, 20 to 45.degree. C., or 25 to 37.degree. C.
The culture period may be, for example, 10 to 120 hours. The
culture may be continued, for example, until the carbon source
contained in the medium is consumed, or until the bacterium loses
the activity. By culturing the bacterium under such conditions as
described above, an L-amino acid is accumulated in cells of the
bacterium and/or the medium.
In the fed-batch culture or continuous culture, feeding of the feed
medium may be continued over the whole period of the culture or
only during a partial period of the culture. In the fed-batch
culture or continuous culture, feeding may be intermittently
performed a plurality of times.
When feeding is intermittently performed a plurality of times, the
feeding may be repeatedly started and stopped so that the period
for one time of feeding is, for example, 30% or shorter, 20% or
shorter, or 10% or shorter, of the total period of the feeding of
the plurality of times.
Furthermore, when feeding is intermittently performed a plurality
of times, the carbon source concentration in the fermentation
medium can also be automatically maintained at a low level by
controlling the feeding so that the second and following feedings
are started when the carbon source in the fermentation medium is
depleted in the non-feeding periods immediately before the
respective feedings (U.S. Pat. No. 5,912,113). Depletion of the
carbon source can be detected on the basis of, for example,
elevation of pH, or elevation of dissolved oxygen
concentration.
In the continuous culture, extraction of the culture medium may be
continued over the whole period of the culture or only during a
partial period of the culture. Furthermore, in the continuous
culture, extraction of the culture medium may be intermittently
performed a plurality of times. Extraction and feeding of the
culture medium may be or may not be simultaneously performed. For
example, after extracting the culture medium, feeding may be
performed, or after performing feeding, the culture medium may be
extracted. It is preferred that the volume of the culture medium to
be extracted is equal to the volume of the medium to be fed. The
expression "the volume of the culture medium to be extracted is
equal to the volume of the medium to be fed equal volume" may mean
that the volume of the culture medium to be extracted is, for
example, 93 to 107% of the volume of the medium to be fed.
When the culture medium is continuously extracted, the extraction
can be started at the same time as or after the start of the
feeding. For example, within 5 hours, 3 hours, or 1 hour, after the
start of the feeding, the extraction can be started.
When the culture medium is intermittently extracted, it is
preferred that, when the concentration of the L-amino acid reaches
a predetermined level, a part of the culture medium is extracted to
collect the L-amino acid, and then a fresh medium is fed to
continue the culture.
Furthermore, after the L-amino acid is collected from the extracted
culture medium, the cells can be reused by recycling filtration
residue containing the cells into the fermentation tank (French
Patent No. 2669935).
Moreover, when L-glutamic acid is produced, the culture can be
performed by using a liquid medium adjusted to satisfy a condition
under which L-glutamic acid is precipitated, while precipitating
L-glutamic acid in the medium. Examples of the condition under
which L-glutamic acid is precipitated include, for example, pH 5.0
to 3.0, pH 4.9 to 3.5, pH 4.9 to 4.0, or around pH 4.7 (EP 1078989
A). The culture may be performed at a pH value within the
aforementioned ranges over the whole period of culture, or only
during a partial period of culture. The term "partial period of
culture" may refer to, for example, 50% or more, 70% or more, 80%
or more, 90% or more, 95% or more, or 99% or more, of the whole
period of culture.
When a basic amino acid such as L-lysine is produced, there may be
employed a method in which the basic amino acid is produced by
fermentation using bicarbonate ions and/or carbonate ions as major
counter ions for the basic amino acid (Japanese Patent Laid-open
(Kokai) No. 2002-65287, U.S. Patent Published Application No.
20020025564, EP 1813677 A). By such a method, a basic amino acid
can be produced while reducing the amounts of sulfate ions and/or
chloride ions to be used, which have been conventionally used as
counter ions for a basic amino acid.
In such a method, pH of the medium is controlled to be 6.5 to 9.0,
6.5 to 8.0, during the culture, and 7.2 to 9.0 at the end of the
culture, so that there is a culture period where 20 mM or more, 30
mM or more, 40 mM or more, of bicarbonate ions and/or carbonate
ions are present in the medium. In order to ensure bicarbonate
and/or carbonate ions exist in the medium in an amount required as
counter ions of the basic amino acid, the internal pressure of the
fermentation tank can be controlled to be positive during the
fermentation, carbon dioxide gas can be supplied into the culture
medium, or both.
The internal pressure of the fermentation tank during fermentation
can be controlled to be positive by, for example, making the gas
supply pressure higher than the exhaust pressure. If the internal
pressure of the fermentation tank is made positive, the carbon
dioxide gas generated by fermentation dissolves in the culture
medium to generate bicarbonate ions and/or carbonate ions, and
these can serve as counter ions of the basic amino acid. The
internal pressure of the fermentation tank can be, specifically,
0.03 to 0.2 MPa, 0.05 to 0.15 MPa, 0.1 to 0.3 MPa, in terms of the
gage pressure (pressure difference with respect to the atmospheric
pressure). When carbon dioxide gas is supplied to the medium, for
example, pure carbon dioxide gas or a mixed gas containing 5 volume
% or more of carbon dioxide gas can be bubbled in the medium. The
internal pressure in the fermentation tank, supply volume of carbon
dioxide gas, and limited aeration volume can be determined by, for
example, measuring pH of the medium, bicarbonate and/or carbonate
ion concentration in the medium, or ammonia concentration in the
medium.
In the conventional methods for producing a basic amino acid, a
sufficient amount of ammonium sulfate and/or ammonium chloride is
usually added to the medium in order to use sulfate ions and/or
chloride ions as counter ions of the basic amino acid, or sulfuric
acid decomposition products and/or hydrochloric acid decomposition
products of proteins etc. are added to the medium as nutrient
components. Therefore, large amounts of sulfate ions and/or
chloride ions are present in the medium, and the concentration of
the weakly acidic carbonate ions is extremely low, i.e., it is at a
ppm order.
On the other hand, the aforementioned method (Japanese Patent
Laid-open (KOKAI) No. 2002-65287, U.S. Patent Published Application
No. 20020025564A, EP 1813677 A) is characterized in that the
amounts of these sulfate ions and chloride ions to be used are
reduced so that the carbon dioxide gas released by microorganism
during fermentation is dissolved in the medium, and used as counter
ions.
That is, to reduce the amounts of sulfate ions and/or chloride ions
to be used is one of the objects of the aforementioned method, and
therefore the total molar concentration of sulfate ions or chloride
ions contained in the medium is usually 700 mM or lower, 500 mM or
lower, 300 mM or lower, 200 mM or lower, or 100 mM or lower. By
lowering the concentrations of sulfate ions and/or chloride ions,
it is easier to ensure the presence of bicarbonate and/or carbonate
ions in the medium. That is, in the aforementioned method, the pH
of the medium to ensure the presence of bicarbonate and/or
carbonate ions in the medium in an amount required as the counter
ions of the basic amino acid can be suppressed to be lower compared
with the conventional methods.
Furthermore, in the aforementioned method, lower concentrations of
anions other than bicarbonate ions and/or carbonate ions (also
referred to as other anions) in the medium are preferred so long as
they are present in amounts required for the growth of the basic
amino acid-producing bacterium. Examples of the other anions
include chloride ions, sulfate ions, phosphate ions, ionized
organic acids, and hydroxide ions. The total molar concentration of
these other anions is usually 900 mM or lower, 700 mM or lower, 500
mM or lower, 300 mM or lower, 200 mM or lower.
In the aforementioned method, it is not necessary to add sulfate
ions or chloride ions to the medium in an amount larger than that
required for growth of the basic amino acid-producing bacterium. It
is preferred that appropriate amounts of ammonium sulfate etc. are
fed to the medium at an early stage of the culture, and the feeding
is terminated in the middle of the culture. Alternatively, ammonium
sulfate etc. may be fed to the medium with maintaining the balance
with respect to the amounts of carbonate ions and/or bicarbonate
ions dissolved in the medium. Furthermore, ammonia may be fed to
the medium as a nitrogen source of the basic amino acid. For
example, when pH is controlled with ammonia, ammonia supplied in
order to elevate pH may be used as a nitrogen source of the basic
amino acid. Ammonia can be supplied to the medium independently or
together with another gas.
In the aforementioned method, it is also preferable to control the
total ammonia concentration in the medium to such a concentration
that production of the basic amino acid is not inhibited. Examples
of such a total ammonia concentration that "production of the basic
amino acid is not inhibited" include, for example, a total ammonia
concentration providing yield and/or productivity corresponding to
50% or more, 70% or more, 90% or more, of the yield and/or
productivity obtainable in the production of the basic amino acid
under optimal conditions. Specifically, for example, the total
ammonia concentration in the medium may be 300 mM or lower, 250 mM
or lower, or 200 mM or lower. The dissociation degree of ammonia
decreases as the pH becomes higher. Non-dissociating ammonia is
more toxic to bacteria compared with ammonium ions. Therefore, the
upper limit of the total ammonia concentration also depends on the
pH of the culture medium. That is, as the pH of the culture medium
increases, the acceptable total ammonia concentration decreases.
Therefore, the total ammonia concentration at which "production of
the basic amino acid is not inhibited" is preferably determined for
each specific pH value. However, the total ammonia concentration
range that is acceptable at the highest pH level during the culture
can be used as the total ammonia concentration range throughout the
entire culture period.
On the other hand, the total concentration of ammonia as a source
of nitrogen required for growth of the basic amino acid-producing
bacterium and production of the basic amino acid is not
particularly limited, and can be appropriately determined, so long
as depletion of ammonia does not continue during the culture, and
decrease in the productivity of the objective substance by the
microorganism due to the shortage of the nitrogen source does not
occur. For example, the ammonia concentration can be measured over
time during the culture, and if ammonia in the medium is depleted,
a small amount of ammonia can be added to the medium. Although the
ammonia concentration after the addition of ammonia is not
particularly limited, the total ammonia concentration can be, for
example, 1 mM or higher, 10 mM or higher, 20 mM or higher.
Furthermore, in the aforementioned method, the medium may contain
cations other than the basic amino acid. Examples of cations other
than the basic amino acid include K, Na, Mg, and Ca originating in
medium components. The total molar concentration of the cations
other than those of the basic amino acid can be 50% or lower of the
molar concentration of the total cations.
Production of the L-amino acid can be confirmed by known methods
used for detection or identification of compounds. Examples of such
methods include, for example, HPLC, LC/MS, GC/MS, and NMR. These
methods can be used in an appropriate combination.
The produced L-amino acid can be collected by known methods used
for separation and purification of compounds. Examples of such
methods include, for example, ion-exchange resin method, membrane
treatment, precipitation, and crystallization. These methods can be
used in an appropriate combination. When the L-amino acid is
accumulated in bacterial cells, the bacterial cells can be
disrupted with, for example, ultrasonic waves or the like, and then
the L-amino acid can be collected by the ion-exchange resin method
or the like from the supernatant obtained by removing the cells
from the cell-disrupted suspension by centrifugation. The L-amino
acid to be collected may be a free compound, a salt thereof, or a
mixture thereof. Examples of the salt include, for example,
sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and
potassium salt. For example, L-lysine may be free L-lysine,
L-lysine sulfate, L-lysine hydrochloride, L-lysine carbonate, or a
mixture of these. Also, for example, L-glutamic acid may be free
L-glutamic acid, sodium L-glutamate (monosodium L-glutamate, MSG),
ammonium L-glutamate (monoammonium L-glutamate), or a mixture of
these. For example, in the case of L-glutamic acid, monosodium
L-glutamate (MSG) can be obtained by adding an acid to the
fermentation broth to crystallize ammonium L-glutamate contained
therein, and then by adding an equimolar of sodium hydroxide to the
crystals. In addition, decolorization can be performed by using
activated carbon before and/or after the crystallization (see,
Tetsuya KAWAKITA, "Industrial Crystallization for Monosodium
L-Glutamate.", Bulletin of the Society of Sea Water Science, Japan,
Vol. 56:5).
When the L-amino acid is precipitated in the medium, it can be
collected by centrifugation, filtration, or the like. The L-amino
acid precipitated in the medium may also be isolated together with
the L-amino acid dissolving in the medium, after the L-amino acid
dissolving in the medium is crystallized.
The collected L-amino acid may contain such components as bacterial
cells, medium components, moisture, and by-product metabolites of
the bacterium in addition to the L-amino acid. The purity of the
collected L-amino acid may be, for example, 30% (w/w) or higher,
50% (w/w) or higher, 70% (w/w) or higher, 80% (w/w) or higher, 90%
(w/w) or higher, or 95% (w/w) or higher.
EXAMPLES
The present invention will be more specifically explained with
reference to the following examples.
Example 1: Impartation of Ethanol-Utilizing Ability to
L-Lysine-Producing Bacterium, AJIK01 Strain (NITE BP-01520)
By using an L-lysine-producing bacterium, Escherichia coli AJIK01
strain (NITE BP-01520), as a parental strain, an L-lysine-producing
bacterium imparted with an ethanol-utilizing ability, AJIK01m2
strain, was constructed.
First, P1 lysate was obtained from the Escherichia coli
MG1655-att-tet-P.sub.L-tacadhE* strain (WO2011/096554) in a
conventional manner, and P1 transduction was performed by using the
AJIK01 strain (NITE BP-01520) as the host to obtain the AJIK01
att-tet-P.sub.L-tacadhE* strain, into which a cassette containing
the adhE* gene was introduced. The adhE* gene is a mutant adhE gene
encoding a mutant AdhE protein corresponding to the wild-type AdhE
protein of the Escherichia coli K-12 MG1655 strain shown as SEQ ID
NO: 46 introduced with six mutations of Glu568Lys, Glu22Gly,
Met236Val, Tyr461Cys, Ile554Ser, and Ala786Val (WO2008/010565).
Then, in order to remove the att-tet sequence introduced upstream
of the P.sub.L-tac promoter, a helper plasmid pMW-intxis-ts (refer
to U.S. Published Patent Application No. 2006/0141586) was used.
The plasmid pMW-intxis-ts is a plasmid carrying a gene encoding the
integrase (Int) of .lamda., phage and a gene encoding excisionase
(Xis) of .lamda., phage, and having temperature-sensitive
replication ability. Competent cells of the AJIK01
att-tet-P.sub.L-tacadhE* strain obtained above were produced in a
conventional manner, transformed with the helper plasmid
pMW-intxis-ts, and cultured at 30.degree. C. on the LB agar medium
containing 100 mg/L of ampicillin, and ampicillin resistant strains
were selected. In order to remove the pMW-intxis-ts plasmid,
transformants were cultured at 42.degree. C. on the LB agar medium.
Ampicillin resistance and tetracycline resistance of the obtained
colonies were examined to obtain a strain sensitive to ampicillin
and tetracycline. The obtained strain is a
P.sub.L-tacadhE*-introduced strain in which the att-tet sequence
was removed from the genome of chromosome, and pMW-intxis-ts was
eliminated. This strain was designated as AJIK01m2 strain.
Example 2: Construction of L-Lysine-Producing Bacterium Having
Enhanced Expression of acnB Gene
(1) Construction of Expression Plasmid
pMW119-attR-Cat-attL-P.sub.14 Containing Promoter P.sub.14
PCR was performed by using the synthetic oligonucleotides shown as
SEQ ID NOS: 2 and 3 as the primers, and the chromosomal DNA of the
Escherichia coli MG1655 strain as the template to amplify the
sequence of the gdhA gene containing the promoter P.sub.14, which
is shown as SEQ ID NO: 1. The PCR product was purified, treated by
using Takara BKL Kit (Takara Bio), and ligated with pMW219 (NIPPON
GENE) digested with SmaI and treated by using Takara BKL Kit to
obtain a plasmid pMW219-P.sub.14-gdhA.
PCR was performed by using the synthetic oligonucleotides shown as
SEQ ID NOS: 4 and 5 as the primers, and pMW219-P.sub.14-gdhA as the
template to amplify the sequence of the promoter P.sub.14 moiety
(P.sub.14 sequence).
PCR was performed by using the synthetic oligonucleotides shown as
SEQ ID NOS: 6 and 7 as the primers, and a plasmid
pMW118-attL-Cm-attR (WO2005/010175) as the template to amplify the
attR-cat-attL sequence having the chloramphenicol resistance gene
cat between the sequences attR and attL of the attachment site of
.lamda. phage.
The attR-cat-attL sequence and P.sub.14 sequence were ligated to
pMW119 (NIPPON GENE) digested with HindIII and SalI by using
In-Fusion HD Cloning Kit (Takara Bio) to construct an expression
plasmid containing the promoter P.sub.14,
pMW119-attR-cat-attL-P.sub.14.
(2) Construction of Plasmid pMW119-attR-Cat-attL-P.sub.14-acnB for
Enhancing Expression of acnB Gene
PCR was performed by using the synthetic oligonucleotides shown as
SEQ ID NOS: 8 and 9 as the primers, and the chromosomal DNA of the
Escherichia coli MG1655 strain as the template to amplify a
sequence containing the acnB gene. PCR was performed by using the
synthetic oligonucleotides shown as SEQ ID NOS: 10 and 11 as the
primers, and the plasmid pMW119-attR-cat-attL-P.sub.14 as the
template to amplify linear pMW119-attR-cat-attL-P.sub.14. The
sequence containing the acnB gene and linear
pMW119-attR-cat-attL-P.sub.14 were ligated by using In-Fusion HD
Cloning Kit (Takara Bio) to construct a plasmid
pMW119-attR-cat-attL-P.sub.14-acnB, which expresses the acnB gene
under the control of the promoter P.sub.14.
The constructed plasmid pMW119-attR-cat-attL-P.sub.14-acnB was
introduced into the AJIK01m2 strain in a conventional manner to
obtain a strain AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnB. The
obtained strain was cultured at 37.degree. C. in the LB medium
containing 100 mg/L of ampicillin until OD600 became about 0.6.
Then, a 40% glycerol solution in the same volume as the culture
broth was added to the culture broth, and the mixture was stirred,
then divided into appropriate volumes, and preserved at -80.degree.
C. This is referred to as glycerol stock of the
AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnB strain.
Example 3: Construction of L-Lysine-Producing Bacterium Having
Enhanced Expression of acnB and aldB Genes
(1) Construction of Plasmid pMW119-attR-Cat-attL-P.sub.14-aldB for
Enhancing expression of aldB gene
PCR was performed by using the synthetic oligonucleotides shown as
SEQ ID NOS: 12 and 13 as the primers, and the chromosomal DNA of
the Escherichia coli MG1655 strain as the template to amplify a
sequence containing the aldB gene. PCR was performed by using the
synthetic oligonucleotides shown as SEQ ID NOS: 10 and 11 as the
primers, and the plasmid pMW119-attR-cat-attL-P.sub.14 as the
template to amplify linear pMW119-attR-cat-attL-P.sub.14. The
sequence containing the aldB gene and linear
pMW119-attR-cat-attL-P.sub.14 were ligated by using In-Fusion HD
Cloning Kit (Takara Bio) to construct a plasmid
pMW119-attR-cat-attL-P.sub.14-aldB, which expresses the aldB gene
under the control of the promoter P.sub.14.
(2) Construction of Plasmid
pMW119-attR-Cat-attL-P.sub.14-acnB-P.sub.14-aldB for Enhancing
Expression of acnB Gene and aldB Gene
PCR was performed by using the synthetic oligonucleotides shown as
SEQ ID NOS: 14 and 15 as the primers, and the plasmid
pMW119-attR-cat-attL-P.sub.14-acnB as the template to amplify a
sequence containing P.sub.14-acnB. PCR was performed by using the
synthetic oligonucleotides shown as SEQ ID NOS: 16 and 17 as the
primers, and the plasmid pMW119-attR-cat-attL-P.sub.14-aldB as the
template to amplify linear pMW119-attR-cat-attL-P.sub.14-aldB. The
sequence containing P.sub.14-acnB and linear
pMW119-attR-cat-attL-P.sub.14-aldB were ligated by using In-Fusion
HD Cloning Kit (Takara Bio) to construct a plasmid
pMW119-attR-cat-attL-P.sub.14-acnB-P.sub.14-aldB, which expresses
the acnB gene and aldB gene under the control of the promoter
P.sub.14.
The constructed plasmid
pMW119-attR-cat-attL-P.sub.14-acnB-P.sub.14-aldB was introduced
into the AJIK01m2 strain in a conventional manner to obtain a
strain AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnB-P.sub.14-aldB.
The obtained strain was cultured at 37.degree. C. in the LB medium
containing 100 mg/L of ampicillin until OD600 became about 0.6.
Then, a 40% glycerol solution of the same volume as the culture
broth was added to the culture broth, and the mixture was stirred,
then divided into appropriate volumes, and preserved at -80.degree.
C. This is referred to as glycerol stock of the
AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnB-P.sub.14-aldB
strain.
Example 4: Construction of L-Lysine-Producing Bacterium Having
Enhanced Expression of acnA and aldB Genes
(1) Construction of Plasmid pMW119-attR-Cat-attL-P.sub.14-acnA for
Enhancing Expression of acnA Gene
PCR was performed by using the synthetic oligonucleotides shown as
SEQ ID NOS: 18 and 19 as the primers, and the chromosomal DNA of
the Escherichia coli MG1655 strain as the template to amplify a
sequence containing the acnA gene. PCR was performed by using the
synthetic oligonucleotides shown as SEQ ID NOS: 10 and 11 as the
primers, and the plasmid pMW119-attR-cat-attL-P.sub.14 as the
template to amplify linear pMW119-attR-cat-attL-P.sub.14. The
sequence containing the acnA gene and linear
pMW119-attR-cat-attL-P.sub.14 were ligated by using In-Fusion HD
Cloning Kit (Takara Bio) to construct a plasmid
pMW119-attR-cat-attL-P.sub.14-acnA, which expresses the acnA gene
under the control of the promoter P.sub.14.
(2) Construction of Plasmid
pMW119-attR-Cat-attL-P.sub.14-acnA-P.sub.14-aldB for Enhancing
Expression of acnA Gene and aldB Gene
PCR was performed by using the synthetic oligonucleotides shown as
SEQ ID NOS: 14 and 20 as the primers, and the plasmid
pMW119-attR-cat-attL-P.sub.14-acnA as the template to amplify a
sequence containing P.sub.14-acnA. PCR was performed by using the
synthetic oligonucleotides shown as SEQ ID NOS: 16 and 17 as the
primers, and the plasmid pMW119-attR-cat-attL-P.sub.14-aldB as the
template to amplify linear pMW119-attR-cat-attL-P.sub.14-aldB. The
sequence containing P.sub.14-acnA and linear
pMW119-attR-cat-attL-P.sub.14-aldB were ligated by using In-Fusion
HD Cloning Kit (Takara Bio) to construct a plasmid
pMW119-attR-cat-attL-P.sub.14-acnA-P.sub.14-aldB, which expresses
the acnA gene and aldB gene under the control of the promoter
P.sub.14.
The constructed plasmid
pMW119-attR-cat-attL-P.sub.14-acnA-P.sub.14-aldB was introduced
into the AJIK01m2 strain in a conventional manner to obtain a
strain AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnA-P.sub.14-aldB.
The obtained strain was cultured at 37.degree. C. in the LB medium
containing 100 mg/L of ampicillin until OD600 became about 0.6.
Then, a 40% glycerol solution in the same volume as the culture
broth was added to the culture broth, and the mixture was stirred,
then divided into appropriate volumes, and preserved at -80.degree.
C. This is referred to as glycerol stock of the
AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnA-P.sub.14-aldB
strain.
Example 5: Evaluation of L-Lysine-Producing Abilities of
L-Lysine-Producing Bacteria
The glycerol stocks obtained in Examples 2, 3, and 4 were each
thawed, and about 100 .mu.L of each was uniformly applied to an L
plate containing 100 mg/L of ampicillin, and incubated at
37.degree. C. for 16 hours as static culture. After the static
culture, the obtained cells were suspended in 0.85% aqueous sodium
chloride, inoculated into 25 mL of a fermentation medium (described
below) containing 100 mg/L of ampicillin contained in a 500
mL-volume Sakaguchi flask so that the turbidity at a wavelength of
600 nm (OD600) became 0.2, and cultured at 37.degree. C. for 24
hours under a condition of stirring at 120 rpm on a reciprocal
shaking culture apparatus. After the shaking culture for 24 hours,
125 .mu.L of ethanol was added to each flask, and shaking culture
was continued for further 17 hours under the same condition.
Composition of the fermentation medium is shown below.
TABLE-US-00001 Ethanol 10 ml/L (NH.sub.4).sub.2SO.sub.4 24 g/L
KH.sub.2PO.sub.4 1.0 g/L MgSO.sub.4.cndot.7H.sub.2O 1.0 g/L
FeSO.sub.4.cndot.7H.sub.2O 0.01 g/L MnSO.sub.4.cndot.5H.sub.2O
0.082 g/L Yeast extract (Difco) 2.0 g/L CaCO.sub.3 (Japanese
Pharmacopoeia) 40 g/L Distilled water To the final volume of 1
L
After the end of the culture, the amount of L-lysine accumulated in
the medium was measured by using Biotech Analyzer AS310 (Sakura
Seiki). Complete consumption of the carbon source (ethanol) added
to the medium was confirmed by using Biotech Analyzer BF-5 (Oji
Scientific Instruments). The amount of the cells at the end of the
culture was measured by measuring the turbidity at a wavelength of
600 nm (OD600) of the culture broth appropriately diluted with 0.2
N dilute hydrochloric acid using a spectrophotometer U-2000
(Hitachi) immediately after the end of the culture.
The results are shown in Table 1. In Table 1, the names of the
strains are mentioned in the column of "Strain", and the amounts of
L-lysine accumulated in the medium are shown in the column of "Lys
(g/L)". The strain having an enhanced expression of the acnB gene
(AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnB strain) showed
significantly higher L-lysine production compared with the control
strain (AJIK01m2/pMW119-attR-cat-attL-P.sub.14 strain). That is, it
was demonstrated that enhancement of the expression of the acnB
gene improves L-lysine-producing ability. Furthermore, the strain
having an enhanced expression of both the acnB and aldB genes
(AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnB-P.sub.14-aldB strain)
showed significantly higher L-lysine production compared with the
control strain (AJIK01m2/pMW119-attR-cat-attL-P.sub.14 strain).
That is, it was demonstrated that simultaneous enhancement of the
expressions of both the acnB gene and aldB gene improves
L-lysine-producing ability. Furthermore, the strain having an
enhanced expression of both the acnA and aldB genes
(AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnA-P.sub.14-aldB strain)
showed significantly higher L-lysine production compared with the
control strain (AJIK01m2/pMW119-attR-cat-attL-P.sub.14 strain).
That is, it was demonstrated that simultaneous enhancement of the
expressions of both the acnA gene and aldB gene improves
L-lysine-producing ability.
TABLE-US-00002 TABLE 1 Evaluation of L-lysine-producing abilities
of L-lysine-producing bacteria Lys Strain OD.sub.600 (g/L)
AJIK01m2/pMW119-attR-cat-attL-P.sub.14 12.1 6.98
AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnB 12.6 7.12
AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnB-P.sub.14-aldB 12.0 7.25
AJIK01m2/pMW119-attR-cat-attL-P.sub.14-acnA-P.sub.14-aldB 13.3
7.25
INDUSTRIAL APPLICABILITY
According to the present invention, an L-amino acid-producing
ability of a bacterium can be improved, and an L-amino acid can be
efficiently produced.
EXPLANATION OF SEQUENCE LISTING
SEQ ID NOS:
1: Nucleotide sequence of promoter P.sub.14
2-20: Primers
21: Nucleotide sequence of acnA gene of Escherichia coli K-12
MG1655
22: Amino acid sequence of AcnA protein of Escherichia coli K-12
MG1655
23: Nucleotide sequence of acnA gene of Pantoea ananatis
AJ13355
24: Amino acid sequence of AcnA protein of Pantoea ananatis
AJ13355
25: Nucleotide sequence of acnA gene of Pectobacterium atrosepticum
SCRI1043
26: Amino acid sequence of AcnA protein of Pectobacterium
atrosepticum SCRI1043
27: Nucleotide sequence of acnA gene of Salmonella enterica serovar
Typhi CT18
28: Amino acid sequence of AcnA protein of Salmonella enterica
serovar Typhi CT18
29: Nucleotide sequence of acnB gene of Escherichia coli K-12
MG1655
30: Amino acid sequence of AcnB protein of Escherichia coli K-12
MG1655
31: Nucleotide sequence of acnB gene of Pantoea ananatis
AJ13355
32: Amino acid sequence of AcnB protein of Pantoea ananatis
AJ13355
33: Nucleotide sequence of acnB gene of Pectobacterium atrosepticum
SCRI1043
34: Amino acid sequence of AcnB protein of Pectobacterium
atrosepticum SCRI1043
35: Nucleotide sequence of acnB gene of Salmonella enterica serovar
Typhi CT18
36: Amino acid sequence of AcnB protein of Salmonella enterica
serovar Typhi CT18
37: Nucleotide sequence of aldB gene of Escherichia coli K-12
MG1655
38: Amino acid sequence of A1 dB protein of Escherichia coli K-12
MG1655
39: Nucleotide sequence of aldB gene of Pantoea ananatis LMG
20103
40: Amino acid sequence of A1 dB protein of Pantoea ananatis LMG
20103
41: Nucleotide sequence of aldB gene of Pectobacterium atrosepticum
SCRI1043
42: Amino acid sequence of A1 dB protein of Pectobacterium
atrosepticum SCRI1043
43: Nucleotide sequence of aldB gene of Salmonella enterica serovar
Typhi CT18
44: Amino acid sequence of A1 dB protein of Salmonella enterica
serovar Typhi CT18
45: Nucleotide sequence of adhE gene of Escherichia coli K-12
MG1655
46: Amino acid sequence of AdhE protein of Escherichia coli K-12
MG1655
47: Nucleotide sequence of adhE gene of Pantoea ananatis LMG
20103
48: Amino acid sequence of AdhE protein of Pantoea ananatis LMG
20103
49: Nucleotide sequence of adhE gene of Pectobacterium atrosepticum
SCRI1043
50: Amino acid sequence of AdhE protein of Pectobacterium
atrosepticum SCRI1043
51: Nucleotide sequence of adhE gene of Salmonella enterica serovar
Typhi CT18
52: Amino acid sequence of AdhE protein of Salmonella enterica
serovar Typhi CT18
SEQUENCE LISTINGS
1
521119DNAArtificial Sequencepromoter P14 1cctgggtcat ttttttcttg
acaaccgtca cattcttgat ggtatagtcg aaaactgcaa 60aagcacatga cataaacaac
ataagcacaa tcgtattaat atataagggt tttatatct 119264DNAArtificial
Sequenceprimer 2gcgggtaccg cggccgccct gggtcatttt tttcttgaca
accgtcacat tcttgatggt 60atag 64347DNAArtificial Sequenceprimer
3gcgcgcgcgc gagctcgcgg ccgctcataa acgcctgaaa ttttgcc
47456DNAArtificial Sequenceprimer 4caggcttcaa gatctcctgg gtcatttttt
tcttgacaac cgtcacattc ttgatg 56567DNAArtificial Sequenceprimer
5atcctctaga gtcgacgcgg ccgctacggc aaaaacgcat cacttcacct tcgctttttc
60ctttcgg 67667DNAArtificial Sequenceprimer 6tgattacgcc aagcttagga
ggttaatcta gacgctcaag ttagtataaa aaagctgaac 60gagaaac
67751DNAArtificial Sequenceprimer 7agatcttgaa gcctgctttt ttatactaag
ttggcattat aaaaaagcat t 51854DNAArtificial Sequenceprimer
8taagggtttt atatctatgc tagaagaata ccgtaagcac gtagctgagc gtgc
54954DNAArtificial Sequenceprimer 9atcctctaga gtcgacttaa accgcagtct
ggaaaatcac cccatcggct ttct 541031DNAArtificial Sequenceprimer
10agatataaaa cccttatata ttaatacgat t 311131DNAArtificial
Sequenceprimer 11gtcgactcta gaggatcccc gggtaccgag c
311256DNAArtificial Sequenceprimer 12taagggtttt atatctatga
ccaataatcc cccttcagca cagattaagc ccggcg 561355DNAArtificial
Sequenceprimer 13atcctctaga gtcgactcag aacagcccca acggtttatc
cgagtagctc accag 551450DNAArtificial Sequenceprimer 14gcaggcttca
agatctcctg ggtcattttt ttcttgacaa ccgtcacatt 501550DNAArtificial
Sequenceprimer 15aaaaaaatga cccaggttaa accgcagtct ggaaaatcac
cccatcggct 501635DNAArtificial Sequenceprimer 16cctgggtcat
ttttttcttg acaaccgtca cattc 351735DNAArtificial Sequenceprimer
17agatcttgaa gcctgctttt ttatactaag ttggc 351849DNAArtificial
Sequenceprimer 18taagggtttt atatctatgt cgtcaaccct acgagaagcc
agtaaggac 491949DNAArtificial Sequenceprimer 19atcctctaga
gtcgacttac ttcaacatat tacgaatgac ataatgcaa 492052DNAArtificial
Sequenceprimer 20aaaaaaatga cccaggttac ttcaacatat tacgaatgac
ataatgcaaa at 52212676DNAEscherichia coli 21atgtcgtcaa ccctacgaga
agccagtaag gacacgttgc aggccaaaga taaaacttac 60cactactaca gcctgccgct
tgctgctaaa tcactgggcg atatcacccg tctacccaag 120tcactcaaag
ttttgctcga aaacctgctg cgctggcagg atggtaactc ggttaccgaa
180gaggatatcc acgcgctggc aggatggctg aaaaatgccc atgctgaccg
tgaaattgcc 240taccgcccgg caagggtgct gatgcaggac tttaccggcg
tacctgccgt tgttgatctg 300gcggcaatgc gcgaagcggt taaacgcctc
ggcggcgata ctgcaaaggt taacccgctc 360tcaccggtcg acctggtcat
tgaccactcg gtgaccgtcg atcgttttgg tgatgatgag 420gcatttgaag
aaaacgtacg cctggaaatg gagcgcaacc acgaacgtta tgtgttcctg
480aaatggggaa agcaagcgtt cagtcggttt agcgtcgtgc cgccaggcac
aggcatttgc 540catcaggtta acctcgaata tctcggcaaa gcagtgtgga
gtgaattgca ggacggtgaa 600tggattgctt atccggatac actcgttggt
actgactcgc acaccaccat gatcaacggc 660cttggcgtgc tggggtgggg
cgttggtggg atcgaagcag aagccgcaat gttaggccag 720ccggtttcca
tgcttatccc ggatgtagtg ggcttcaaac ttaccggaaa attacgtgaa
780ggtattaccg ccacagacct ggttctcact gttacccaaa tgctgcgcaa
acatggcgtg 840gtggggaaat tcgtcgaatt ttatggtgat ggtctggatt
cactaccgtt ggcggatcgc 900gccaccattg ccaatatgtc gccagaatat
ggtgccacct gtggcttctt cccaatcgat 960gctgtaaccc tcgattacat
gcgtttaagc gggcgcagcg aagatcaggt cgagttggtc 1020gaaaaatatg
ccaaagcgca gggcatgtgg cgtaacccgg gcgatgaacc aatttttacc
1080agtacgttag aactggatat gaatgacgtt gaagcgagcc tggcagggcc
taaacgccca 1140caggatcgcg ttgcactgcc cgatgtacca aaagcatttg
ccgccagtaa cgaactggaa 1200gtgaatgcca cgcataaaga tcgccagccg
gtcgattatg ttatgaacgg acatcagtat 1260cagttacctg atggcgctgt
ggtcattgct gcgataacct cgtgcaccaa cacctctaac 1320ccaagtgtgc
tgatggccgc aggcttgctg gcgaaaaaag ccgtaactct gggcctcaag
1380cggcaaccat gggtcaaagc gtcgctggca ccgggttcga aagtcgtttc
tgattatctg 1440gcaaaagcga aactgacacc gtatctcgac gaactggggt
ttaaccttgt gggatacggt 1500tgtaccacct gtattggtaa ctctgggccg
ctgcccgatc ctatcgaaac ggcaatcaaa 1560aaaagcgatt taaccgtcgg
tgcggtgctg tccggcaacc gtaactttga aggccgtatc 1620catccgctgg
ttaaaactaa ctggctggcc tcgccgccgc tggtggttgc ctatgcgctg
1680gcgggaaata tgaatatcaa cctggcttct gagcctatcg gccatgatcg
caaaggcgat 1740ccggtttatc tgaaagatat ctggccatcg gcacaagaaa
ttgcccgtgc ggtagaacaa 1800gtctccacag aaatgttccg caaagagtac
gcagaagttt ttgaaggcac agcagagtgg 1860aagggaatta acgtcacacg
atccgatacc tacggttggc aggaggactc aacctatatt 1920cgcttatcgc
ctttctttga tgaaatgcag gcaacaccag caccagtgga agatattcac
1980ggtgcgcgga tcctcgcaat gctgggggat tcagtcacca ctgaccatat
ctctccggcg 2040ggcagtatta agcccgacag cccagcgggt cgatatctac
aaggtcgggg tgttgagcga 2100aaagacttta actcctacgg ttcgcggcgt
ggtaaccatg aagtgatgat gcgcggcacc 2160ttcgccaata ttcgcatccg
taatgaaatg gtgcctggcg ttgaaggggg gatgacgcgg 2220catttacctg
acagcgacgt agtctctatt tatgatgctg cgatgcgcta taagcaggag
2280caaacgccgc tggcggtgat tgccgggaaa gagtatggat caggctccag
tcgtgactgg 2340gcggcaaaag gtccgcgtct gcttggtatt cgtgtggtga
ttgccgaatc gtttgaacga 2400attcaccgtt cgaatttaat tggcatgggc
atcctgccgc tggaatttcc gcaaggcgta 2460acgcgtaaaa cgttagggct
aaccggggaa gagaagattg atattggcga tctgcaaaac 2520ctacaacccg
gcgcgacggt tccggtgacg cttacgcgcg cggatggtag ccaggaagtc
2580gtaccctgcc gttgtcgtat cgacaccgcg acggagttga cctactacca
gaacgacggc 2640attttgcatt atgtcattcg taatatgttg aagtaa
267622891PRTEscherichia coli 22Met Ser Ser Thr Leu Arg Glu Ala Ser
Lys Asp Thr Leu Gln Ala Lys1 5 10 15Asp Lys Thr Tyr His Tyr Tyr Ser
Leu Pro Leu Ala Ala Lys Ser Leu 20 25 30Gly Asp Ile Thr Arg Leu Pro
Lys Ser Leu Lys Val Leu Leu Glu Asn 35 40 45Leu Leu Arg Trp Gln Asp
Gly Asn Ser Val Thr Glu Glu Asp Ile His 50 55 60Ala Leu Ala Gly Trp
Leu Lys Asn Ala His Ala Asp Arg Glu Ile Ala65 70 75 80Tyr Arg Pro
Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala 85 90 95Val Val
Asp Leu Ala Ala Met Arg Glu Ala Val Lys Arg Leu Gly Gly 100 105
110Asp Thr Ala Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125His Ser Val Thr Val Asp Arg Phe Gly Asp Asp Glu Ala Phe
Glu Glu 130 135 140Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg
Tyr Val Phe Leu145 150 155 160Lys Trp Gly Lys Gln Ala Phe Ser Arg
Phe Ser Val Val Pro Pro Gly 165 170 175Thr Gly Ile Cys His Gln Val
Asn Leu Glu Tyr Leu Gly Lys Ala Val 180 185 190Trp Ser Glu Leu Gln
Asp Gly Glu Trp Ile Ala Tyr Pro Asp Thr Leu 195 200 205Val Gly Thr
Asp Ser His Thr Thr Met Ile Asn Gly Leu Gly Val Leu 210 215 220Gly
Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln225 230
235 240Pro Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr
Gly 245 250 255Lys Leu Arg Glu Gly Ile Thr Ala Thr Asp Leu Val Leu
Thr Val Thr 260 265 270Gln Met Leu Arg Lys His Gly Val Val Gly Lys
Phe Val Glu Phe Tyr 275 280 285Gly Asp Gly Leu Asp Ser Leu Pro Leu
Ala Asp Arg Ala Thr Ile Ala 290 295 300Asn Met Ser Pro Glu Tyr Gly
Ala Thr Cys Gly Phe Phe Pro Ile Asp305 310 315 320Ala Val Thr Leu
Asp Tyr Met Arg Leu Ser Gly Arg Ser Glu Asp Gln 325 330 335Val Glu
Leu Val Glu Lys Tyr Ala Lys Ala Gln Gly Met Trp Arg Asn 340 345
350Pro Gly Asp Glu Pro Ile Phe Thr Ser Thr Leu Glu Leu Asp Met Asn
355 360 365Asp Val Glu Ala Ser Leu Ala Gly Pro Lys Arg Pro Gln Asp
Arg Val 370 375 380Ala Leu Pro Asp Val Pro Lys Ala Phe Ala Ala Ser
Asn Glu Leu Glu385 390 395 400Val Asn Ala Thr His Lys Asp Arg Gln
Pro Val Asp Tyr Val Met Asn 405 410 415Gly His Gln Tyr Gln Leu Pro
Asp Gly Ala Val Val Ile Ala Ala Ile 420 425 430Thr Ser Cys Thr Asn
Thr Ser Asn Pro Ser Val Leu Met Ala Ala Gly 435 440 445Leu Leu Ala
Lys Lys Ala Val Thr Leu Gly Leu Lys Arg Gln Pro Trp 450 455 460Val
Lys Ala Ser Leu Ala Pro Gly Ser Lys Val Val Ser Asp Tyr Leu465 470
475 480Ala Lys Ala Lys Leu Thr Pro Tyr Leu Asp Glu Leu Gly Phe Asn
Leu 485 490 495Val Gly Tyr Gly Cys Thr Thr Cys Ile Gly Asn Ser Gly
Pro Leu Pro 500 505 510Asp Pro Ile Glu Thr Ala Ile Lys Lys Ser Asp
Leu Thr Val Gly Ala 515 520 525Val Leu Ser Gly Asn Arg Asn Phe Glu
Gly Arg Ile His Pro Leu Val 530 535 540Lys Thr Asn Trp Leu Ala Ser
Pro Pro Leu Val Val Ala Tyr Ala Leu545 550 555 560Ala Gly Asn Met
Asn Ile Asn Leu Ala Ser Glu Pro Ile Gly His Asp 565 570 575Arg Lys
Gly Asp Pro Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln 580 585
590Glu Ile Ala Arg Ala Val Glu Gln Val Ser Thr Glu Met Phe Arg Lys
595 600 605Glu Tyr Ala Glu Val Phe Glu Gly Thr Ala Glu Trp Lys Gly
Ile Asn 610 615 620Val Thr Arg Ser Asp Thr Tyr Gly Trp Gln Glu Asp
Ser Thr Tyr Ile625 630 635 640Arg Leu Ser Pro Phe Phe Asp Glu Met
Gln Ala Thr Pro Ala Pro Val 645 650 655Glu Asp Ile His Gly Ala Arg
Ile Leu Ala Met Leu Gly Asp Ser Val 660 665 670Thr Thr Asp His Ile
Ser Pro Ala Gly Ser Ile Lys Pro Asp Ser Pro 675 680 685Ala Gly Arg
Tyr Leu Gln Gly Arg Gly Val Glu Arg Lys Asp Phe Asn 690 695 700Ser
Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr705 710
715 720Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val Pro Gly Val Glu
Gly 725 730 735Gly Met Thr Arg His Leu Pro Asp Ser Asp Val Val Ser
Ile Tyr Asp 740 745 750Ala Ala Met Arg Tyr Lys Gln Glu Gln Thr Pro
Leu Ala Val Ile Ala 755 760 765Gly Lys Glu Tyr Gly Ser Gly Ser Ser
Arg Asp Trp Ala Ala Lys Gly 770 775 780Pro Arg Leu Leu Gly Ile Arg
Val Val Ile Ala Glu Ser Phe Glu Arg785 790 795 800Ile His Arg Ser
Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe 805 810 815Pro Gln
Gly Val Thr Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Lys 820 825
830Ile Asp Ile Gly Asp Leu Gln Asn Leu Gln Pro Gly Ala Thr Val Pro
835 840 845Val Thr Leu Thr Arg Ala Asp Gly Ser Gln Glu Val Val Pro
Cys Arg 850 855 860Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr
Gln Asn Asp Gly865 870 875 880Ile Leu His Tyr Val Ile Arg Asn Met
Leu Lys 885 890232682DNAPantoea ananatis 23atgtcgtcaa ccctacgcga
gcagagtcag gaaacactgc aggtagataa tcaaaactat 60cacatcttca gtctacctcg
cgcatcacaa catctcggca acattgatcg tttgcctaaa 120tccatgaaag
ttctgctgga aaacctgctg cgctggcagg acggtgattc agtcacggaa
180gaggatatcc aggcactggt cgactggcag aaaacggccc atggcgatcg
ggaaattgcc 240tatcgccctg cgcgtgtatt aatgcaggac ttcactggcg
tgcctgccgt ggtggatttg 300gccgccatgc gtgaggcggt gagtcgtctt
ggcggcgacg tcgccaaagt gaatcctctg 360tcgccggtcg atctggttat
cgaccactct gtcacggttg accatttcgg cgacgataac 420gcatttgaag
aaaacgtgcg gctggaaatg gagcgcaacc atgaacgcta cgtttttctg
480cgctggggcc aaaaggcttt tgatcagttt cgggtggtac cgccaggcac
aggtatctgt 540catcaggtga acctggaata cctgggcaaa gcgatctggc
agcagaacat taacggtgaa 600cgttacgcct ggcctgatac gttagtcgga
accgattctc ataccaccat gatcaatgcc 660ctgggtgtac tcggctgggg
cgtgggcggg attgaggcag aggccgccat gcttggccag 720ccggtttcta
tgctgatccc cgacgtcgtg ggtttcaagc taaccgggaa actgcgtgcg
780ggcatcaccg caaccgacct tgtacttacc gtcacgcaaa tgctgcgtaa
gcacggcgtt 840gtcgggaagt ttgtcgagtt ctatggcgat ggcctggctg
atctgccgct ggccgaccgt 900gccaccattg ctaatatggc accagaatat
ggcgcaacct gcggattttt cccggttgat 960gaagttacac tcagctatat
gacgctaacc ggacgcgatg ccgagcaggt ggcactggtt 1020gaacactatg
ctaagcgaca gggcctgtgg cgtaatgcgg gcgatgaacc gattttcacc
1080agtagccttg cgctcgatat gaatgaagtc gagtcgagcc tggccggacc
gaaacgtccg 1140caggatcgcg tctcgctggg ggatgtgccc gccgctttcg
atgccagcaa tgagctggaa 1200gtgaaccatg cacagaaacc gcataagcag
gtcgactata ccgacagcga gaccggcctg 1260agccacacgc tgaccgatgg
cgcggtggcg attgcggcga ttacctcctg taccaacacc 1320tctaacccca
gcgtgctgat ggccgcaggg ttactggcga aaaaagcggt tgagcgggga
1380ttaaaacgcc agccgtgggt caaagcgtcg ctggctccgg gttctaaagt
ggtctccgac 1440tatctcgcgg tcgcgcagct gacacctcat ctcgataaac
tgggttttaa ccttgtgggc 1500tacggttgta cgacctgtat cggtaactcg
ggtccgctgc cggatgagat tgaatcggcc 1560atcaaagaag gggatttaac
ggtaggggcc gtcctgtccg gtaaccgcaa cttcgagggc 1620cgtattcatc
cgctgattaa aaccaactgg ctggcatcgc caccgctggt cgtcgcctat
1680gcgctggcag gtaatatgaa aatcaacctg caaaccgacc cgctcggtca
cgatcatcag 1740ggcaagccgg tatttctgaa agacatctgg ccttcacctg
aagagattgc gaccgccgtc 1800cagcaggtca ccagcgatat gtatcacaaa
gagtacgctg aggtatttaa cggcacgcca 1860gagtggcagg ccattaaggt
aagtgaagcg gctacctacg actgggatga aggatcaacc 1920tacattcgcc
tgtcgccctt ctttgacgac atggaaaaag aacctaagcc ggttcaggat
1980attcatggcg cgcgcctgct ggcgattctt ggcgattcgg tcaccactga
ccacatctcg 2040cctgccggta gcatcaaagc ggaaagtccg gccggtcgct
atctgctgtc ccacggcgtg 2100gagcggaatg attttaactc ctacggttca
cggcgcggca accacgaagt gatgatgcgc 2160gggacctttg cgaatatccg
tattcgaaat gaaatggtgc ccggcgtgga agggggttac 2220accaaacact
accccagcgg tgagcagttg gccatttatg acgcggcgat gaaatatcag
2280gctgacggca ttcccctggc ggtgattgcg ggtaaagagt acggttcagg
atcgagccgt 2340gactgggcgg cgaaagggcc gcgtttgcaa ggcgtccgcg
tggtaattgc ggaatccttc 2400gaacgtattc accgttccaa cctgattggt
atggggattt tgccgctgga gttccctcag 2460ggcgtaacgc gtaaaacgtt
aggtctgaaa ggggatgagg cgatagatgt ggaaaacctg 2520gcgcagctta
aacccggatg caccgtttct gtcacgctaa cgcgcgcaga tggcagtcag
2580gagaagctgg atacgcgttg ccgtattgat accggtaatg aactgaccta
ttaccgaaac 2640gacggtattc tgcactacgt gattcgtaac atgctgaact ga
268224893PRTPantoea ananatis 24Met Ser Ser Thr Leu Arg Glu Gln Ser
Gln Glu Thr Leu Gln Val Asp1 5 10 15Asn Gln Asn Tyr His Ile Phe Ser
Leu Pro Arg Ala Ser Gln His Leu 20 25 30Gly Asn Ile Asp Arg Leu Pro
Lys Ser Met Lys Val Leu Leu Glu Asn 35 40 45Leu Leu Arg Trp Gln Asp
Gly Asp Ser Val Thr Glu Glu Asp Ile Gln 50 55 60Ala Leu Val Asp Trp
Gln Lys Thr Ala His Gly Asp Arg Glu Ile Ala65 70 75 80Tyr Arg Pro
Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala 85 90 95Val Val
Asp Leu Ala Ala Met Arg Glu Ala Val Ser Arg Leu Gly Gly 100 105
110Asp Val Ala Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125His Ser Val Thr Val Asp His Phe Gly Asp Asp Asn Ala Phe
Glu Glu 130 135 140Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg
Tyr Val Phe Leu145 150 155 160Arg Trp Gly Gln Lys Ala Phe Asp Gln
Phe Arg Val Val Pro Pro Gly 165 170 175Thr Gly Ile Cys His Gln Val
Asn Leu Glu Tyr Leu Gly Lys Ala Ile 180 185 190Trp Gln Gln Asn Ile
Asn Gly Glu Arg Tyr Ala Trp Pro Asp Thr Leu 195 200 205Val Gly Thr
Asp Ser His Thr Thr Met Ile Asn Ala Leu Gly Val Leu 210 215 220Gly
Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln225 230
235 240Pro Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr
Gly 245 250 255Lys Leu Arg Ala Gly Ile Thr Ala Thr Asp Leu Val Leu
Thr Val Thr 260 265 270Gln Met Leu Arg Lys His Gly Val Val Gly Lys
Phe Val Glu Phe Tyr 275 280 285Gly Asp Gly Leu Ala Asp Leu Pro Leu
Ala Asp Arg Ala Thr Ile Ala 290 295 300Asn Met Ala Pro Glu Tyr Gly
Ala Thr Cys Gly Phe Phe Pro Val Asp305 310 315 320Glu Val Thr Leu
Ser Tyr Met Thr Leu Thr Gly Arg Asp Ala Glu Gln 325 330 335Val Ala
Leu Val Glu His Tyr Ala Lys Arg Gln Gly Leu Trp Arg Asn 340
345 350Ala Gly Asp Glu Pro Ile Phe Thr Ser Ser Leu Ala Leu Asp Met
Asn 355 360 365Glu Val Glu Ser Ser Leu Ala Gly Pro Lys Arg Pro Gln
Asp Arg Val 370 375 380Ser Leu Gly Asp Val Pro Ala Ala Phe Asp Ala
Ser Asn Glu Leu Glu385 390 395 400Val Asn His Ala Gln Lys Pro His
Lys Gln Val Asp Tyr Thr Asp Ser 405 410 415Glu Thr Gly Leu Ser His
Thr Leu Thr Asp Gly Ala Val Ala Ile Ala 420 425 430Ala Ile Thr Ser
Cys Thr Asn Thr Ser Asn Pro Ser Val Leu Met Ala 435 440 445Ala Gly
Leu Leu Ala Lys Lys Ala Val Glu Arg Gly Leu Lys Arg Gln 450 455
460Pro Trp Val Lys Ala Ser Leu Ala Pro Gly Ser Lys Val Val Ser
Asp465 470 475 480Tyr Leu Ala Val Ala Gln Leu Thr Pro His Leu Asp
Lys Leu Gly Phe 485 490 495Asn Leu Val Gly Tyr Gly Cys Thr Thr Cys
Ile Gly Asn Ser Gly Pro 500 505 510Leu Pro Asp Glu Ile Glu Ser Ala
Ile Lys Glu Gly Asp Leu Thr Val 515 520 525Gly Ala Val Leu Ser Gly
Asn Arg Asn Phe Glu Gly Arg Ile His Pro 530 535 540Leu Ile Lys Thr
Asn Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr545 550 555 560Ala
Leu Ala Gly Asn Met Lys Ile Asn Leu Gln Thr Asp Pro Leu Gly 565 570
575His Asp His Gln Gly Lys Pro Val Phe Leu Lys Asp Ile Trp Pro Ser
580 585 590Pro Glu Glu Ile Ala Thr Ala Val Gln Gln Val Thr Ser Asp
Met Tyr 595 600 605His Lys Glu Tyr Ala Glu Val Phe Asn Gly Thr Pro
Glu Trp Gln Ala 610 615 620Ile Lys Val Ser Glu Ala Ala Thr Tyr Asp
Trp Asp Glu Gly Ser Thr625 630 635 640Tyr Ile Arg Leu Ser Pro Phe
Phe Asp Asp Met Glu Lys Glu Pro Lys 645 650 655Pro Val Gln Asp Ile
His Gly Ala Arg Leu Leu Ala Ile Leu Gly Asp 660 665 670Ser Val Thr
Thr Asp His Ile Ser Pro Ala Gly Ser Ile Lys Ala Glu 675 680 685Ser
Pro Ala Gly Arg Tyr Leu Leu Ser His Gly Val Glu Arg Asn Asp 690 695
700Phe Asn Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met
Arg705 710 715 720Gly Thr Phe Ala Asn Ile Arg Ile Arg Asn Glu Met
Val Pro Gly Val 725 730 735Glu Gly Gly Tyr Thr Lys His Tyr Pro Ser
Gly Glu Gln Leu Ala Ile 740 745 750Tyr Asp Ala Ala Met Lys Tyr Gln
Ala Asp Gly Ile Pro Leu Ala Val 755 760 765Ile Ala Gly Lys Glu Tyr
Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala 770 775 780Lys Gly Pro Arg
Leu Gln Gly Val Arg Val Val Ile Ala Glu Ser Phe785 790 795 800Glu
Arg Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu 805 810
815Glu Phe Pro Gln Gly Val Thr Arg Lys Thr Leu Gly Leu Lys Gly Asp
820 825 830Glu Ala Ile Asp Val Glu Asn Leu Ala Gln Leu Lys Pro Gly
Cys Thr 835 840 845Val Ser Val Thr Leu Thr Arg Ala Asp Gly Ser Gln
Glu Lys Leu Asp 850 855 860Thr Arg Cys Arg Ile Asp Thr Gly Asn Glu
Leu Thr Tyr Tyr Arg Asn865 870 875 880Asp Gly Ile Leu His Tyr Val
Ile Arg Asn Met Leu Asn 885 890252673DNAPectobacterium atrosepticum
25atgtcatcac accttcgcga cacttgtctg gacacactga cggttcgaca gcagatttac
60cattactaca gcctgccgaa ggcggcgaaa acgcttggca atatcgataa attaccgaag
120tcactcaagg tattgctgga aaatttattg cgtcatcagg acggcgacac
ggtcgagcag 180gacgatcttc aggcggtcgt ggactggctg aaaatcggtc
acgccgatcg ggaaatcgcc 240tatcggccag cgcgcgtact gatgcaggac
tttaccggcg tgcccgccgt ggtcgatctg 300gcggcgatgc gtgcagcggt
gaaacggttg ggcggcgatg ttaataaggt caacccgctg 360tcgccggtcg
atctggttat tgaccactcg gttacggttg atcacttcgg cgatcgtcag
420gcgctagcgg ataacacgca gttggaaatg gcgcgtaacc gtgaacgtta
tgagtttttg 480cgctggggac aaaatgcctt tagccacttc agcgtcgtgc
cgccgggaac cgggatctgc 540catcaggtga atctggagta tctcgccaag
gccatctggt acgaaaaaca gggcgacaaa 600cagtttgcct accctgatac
gctggtagga accgattcgc acaccaccat gattaacggt 660ttgggcgtgc
tcggctgggg tgtcggtggg atagaagcag aagccgcgat gttggggcaa
720cctgtttcga tgctgattcc cgatgtggtt ggcgtcaaat taagcggcaa
aatgcaagaa 780gggatcacgg caaccgattt ggttctgacg gtaacgcaga
tgctgcgtaa acacggcgtt 840gtcggcaaat ttgtggaatt ttacggtgat
gggctggatt ctctaccgtt ggcagatcgc 900gcgactatcg ccaacatggc
accggaatat ggtgcaacct gtggcttttt ccctatcgat 960cacatcacgc
tggattacat gcggttgacc aaccgcgccg aagaacagat tgcactggtg
1020gaagcctaca gtaagcaaca ggggctgtgg cgcaatgctg gagatgagcc
ggtattcacc 1080agccagctag cgctggattt ggcaacggtg gaaaccagtc
tggcagggcc gaaacgccca 1140caggatcgcg tgcctttagc gggtgtgccg
gaagccttta aagccagccg ggaactggat 1200gtcagctcgg tgaagaaccg
ctctgactat gaagagttca cgttggaagg tgagacgcat 1260cgcttgcatc
agggggcggt cgtgatcgcg gcgatcacct cttgtactaa tacctccaac
1320cctagcgtgc tgatgacggc tgggctactg gcaaaaaatg ccgtagagcg
tggtcttaaa 1380accaagccgt gggtcaaaac ctcgttggca ccgggctcac
gggtcgtcac ggattactat 1440gctaaagcgg aattaacgcc atacctcgac
gagttggggt tcaatctggt ggggtacggc 1500tgtaccacct gtatcggtaa
ctccggcccg ctgccggatg cgattgaagc cgcgataaaa 1560gaaggcgact
tgacggtcgg cgccgtgttg tcaggtaacc gtaactttga aggccgtatt
1620catccgctgg tgaagactaa ctggctggcg tcaccgccgc tggtggtcgc
ctacgcgctg 1680gcgggaaata tgaatgtcga tctgacgcaa caaccgctgg
gtgaagatcg tgacggaaaa 1740gccgtttatc tgaaagacat ctggccctcg
acgaaagcgg tggcggacgc ggtattgaat 1800gtcaacgctg gcatgttcca
caaacaatat gccgcagtgt ttgaaggtac gcaggagtgg 1860caagatatcg
aggtcgacga taatcctacc tatcagtggc cggaagaatc gacctatatt
1920cgccagacgc ctttctttct ggatatgggg aaagaaccgg agccggttca
ggatatccac 1980aaggcgcgca ttctggcgat gctgggcgat tcggttacaa
ccgaccacat ctcgccagca 2040ggcaacatca agcgtgatag ccctgcaggg
aaatatttgc tggaacgcgg cgttgaaacc 2100acggagttca actcttacgg
ttcacggcgg ggcaaccacg aagtgatgat gcgcgggacg 2160tttgccaaca
tccgtatccg taatgaaatg gtgccgggta aagagggcgg ttatacccgt
2220cacattccgt cgcagaatga gatgacgatc tatgacgcgg caatgcgcta
caaggaagaa 2280ggtgtctcac tggcgctgtt tgctgggaaa gagtacggtt
cgggttctag ccgagactgg 2340gccgcgaaag gcccacgttt gctgggtgtt
cgcgtggtga tcgccgaatc gttcgagcgt 2400attcaccgct ctaacctgat
tgggatgggg attctgccgc tggaatttcc cgacggtgtg 2460acgcgtaaaa
cgctgcaatt aaccggggat gagcagattt cgattacggg attaaatcaa
2520ctggcacctg gcgccacggt tgaggtaaat atcacggatg ctgatggtaa
tacgcaggtg 2580atcaacaccc gctgccgcat cgacacccgt aatgaactga
cctattacca gaacgacggt 2640atccttcatt acgttatccg taatatgttg taa
267326890PRTPectobacterium atrosepticum 26Met Ser Ser His Leu Arg
Asp Thr Cys Leu Asp Thr Leu Thr Val Arg1 5 10 15Gln Gln Ile Tyr His
Tyr Tyr Ser Leu Pro Lys Ala Ala Lys Thr Leu 20 25 30Gly Asn Ile Asp
Lys Leu Pro Lys Ser Leu Lys Val Leu Leu Glu Asn 35 40 45Leu Leu Arg
His Gln Asp Gly Asp Thr Val Glu Gln Asp Asp Leu Gln 50 55 60Ala Val
Val Asp Trp Leu Lys Ile Gly His Ala Asp Arg Glu Ile Ala65 70 75
80Tyr Arg Pro Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala
85 90 95Val Val Asp Leu Ala Ala Met Arg Ala Ala Val Lys Arg Leu Gly
Gly 100 105 110Asp Val Asn Lys Val Asn Pro Leu Ser Pro Val Asp Leu
Val Ile Asp 115 120 125His Ser Val Thr Val Asp His Phe Gly Asp Arg
Gln Ala Leu Ala Asp 130 135 140Asn Thr Gln Leu Glu Met Ala Arg Asn
Arg Glu Arg Tyr Glu Phe Leu145 150 155 160Arg Trp Gly Gln Asn Ala
Phe Ser His Phe Ser Val Val Pro Pro Gly 165 170 175Thr Gly Ile Cys
His Gln Val Asn Leu Glu Tyr Leu Ala Lys Ala Ile 180 185 190Trp Tyr
Glu Lys Gln Gly Asp Lys Gln Phe Ala Tyr Pro Asp Thr Leu 195 200
205Val Gly Thr Asp Ser His Thr Thr Met Ile Asn Gly Leu Gly Val Leu
210 215 220Gly Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu
Gly Gln225 230 235 240Pro Val Ser Met Leu Ile Pro Asp Val Val Gly
Val Lys Leu Ser Gly 245 250 255Lys Met Gln Glu Gly Ile Thr Ala Thr
Asp Leu Val Leu Thr Val Thr 260 265 270Gln Met Leu Arg Lys His Gly
Val Val Gly Lys Phe Val Glu Phe Tyr 275 280 285Gly Asp Gly Leu Asp
Ser Leu Pro Leu Ala Asp Arg Ala Thr Ile Ala 290 295 300Asn Met Ala
Pro Glu Tyr Gly Ala Thr Cys Gly Phe Phe Pro Ile Asp305 310 315
320His Ile Thr Leu Asp Tyr Met Arg Leu Thr Asn Arg Ala Glu Glu Gln
325 330 335Ile Ala Leu Val Glu Ala Tyr Ser Lys Gln Gln Gly Leu Trp
Arg Asn 340 345 350Ala Gly Asp Glu Pro Val Phe Thr Ser Gln Leu Ala
Leu Asp Leu Ala 355 360 365Thr Val Glu Thr Ser Leu Ala Gly Pro Lys
Arg Pro Gln Asp Arg Val 370 375 380Pro Leu Ala Gly Val Pro Glu Ala
Phe Lys Ala Ser Arg Glu Leu Asp385 390 395 400Val Ser Ser Val Lys
Asn Arg Ser Asp Tyr Glu Glu Phe Thr Leu Glu 405 410 415Gly Glu Thr
His Arg Leu His Gln Gly Ala Val Val Ile Ala Ala Ile 420 425 430Thr
Ser Cys Thr Asn Thr Ser Asn Pro Ser Val Leu Met Thr Ala Gly 435 440
445Leu Leu Ala Lys Asn Ala Val Glu Arg Gly Leu Lys Thr Lys Pro Trp
450 455 460Val Lys Thr Ser Leu Ala Pro Gly Ser Arg Val Val Thr Asp
Tyr Tyr465 470 475 480Ala Lys Ala Glu Leu Thr Pro Tyr Leu Asp Glu
Leu Gly Phe Asn Leu 485 490 495Val Gly Tyr Gly Cys Thr Thr Cys Ile
Gly Asn Ser Gly Pro Leu Pro 500 505 510Asp Ala Ile Glu Ala Ala Ile
Lys Glu Gly Asp Leu Thr Val Gly Ala 515 520 525Val Leu Ser Gly Asn
Arg Asn Phe Glu Gly Arg Ile His Pro Leu Val 530 535 540Lys Thr Asn
Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Leu545 550 555
560Ala Gly Asn Met Asn Val Asp Leu Thr Gln Gln Pro Leu Gly Glu Asp
565 570 575Arg Asp Gly Lys Ala Val Tyr Leu Lys Asp Ile Trp Pro Ser
Thr Lys 580 585 590Ala Val Ala Asp Ala Val Leu Asn Val Asn Ala Gly
Met Phe His Lys 595 600 605Gln Tyr Ala Ala Val Phe Glu Gly Thr Gln
Glu Trp Gln Asp Ile Glu 610 615 620Val Asp Asp Asn Pro Thr Tyr Gln
Trp Pro Glu Glu Ser Thr Tyr Ile625 630 635 640Arg Gln Thr Pro Phe
Phe Leu Asp Met Gly Lys Glu Pro Glu Pro Val 645 650 655Gln Asp Ile
His Lys Ala Arg Ile Leu Ala Met Leu Gly Asp Ser Val 660 665 670Thr
Thr Asp His Ile Ser Pro Ala Gly Asn Ile Lys Arg Asp Ser Pro 675 680
685Ala Gly Lys Tyr Leu Leu Glu Arg Gly Val Glu Thr Thr Glu Phe Asn
690 695 700Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg
Gly Thr705 710 715 720Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val
Pro Gly Lys Glu Gly 725 730 735Gly Tyr Thr Arg His Ile Pro Ser Gln
Asn Glu Met Thr Ile Tyr Asp 740 745 750Ala Ala Met Arg Tyr Lys Glu
Glu Gly Val Ser Leu Ala Leu Phe Ala 755 760 765Gly Lys Glu Tyr Gly
Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly 770 775 780Pro Arg Leu
Leu Gly Val Arg Val Val Ile Ala Glu Ser Phe Glu Arg785 790 795
800Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe
805 810 815Pro Asp Gly Val Thr Arg Lys Thr Leu Gln Leu Thr Gly Asp
Glu Gln 820 825 830Ile Ser Ile Thr Gly Leu Asn Gln Leu Ala Pro Gly
Ala Thr Val Glu 835 840 845Val Asn Ile Thr Asp Ala Asp Gly Asn Thr
Gln Val Ile Asn Thr Arg 850 855 860Cys Arg Ile Asp Thr Arg Asn Glu
Leu Thr Tyr Tyr Gln Asn Asp Gly865 870 875 880Ile Leu His Tyr Val
Ile Arg Asn Met Leu 885 890272676DNASalmonella enterica
27atgtcgtcaa ccctacgaga agccagtaag gatacattgc aggccaaaga taaaacgtat
60cattactaca gtctgccgct ggctgccaaa tccctgggcg atatcgcccg tttgcccaaa
120tcacttaaag tgttactgga aaacctgttg cgctggcagg acggcgaatc
tgtgactgat 180gaagatattc aggcgctggc cggttggctt aaaaatgccc
atgccgatcg tgaaattgct 240tggcggcccg cccgtgtcct gatgcaggac
tttaccggcg tgcctgccgt tgtcgacctg 300gcggcgatgc gtgaagccgt
taaacgcctc ggcggcgata cgtcgaaagt gaacccgtta 360tcgccggttg
atctggttat tgaccactct gtgacggtcg atcatttcgg cgatgatgat
420gcgtttgaag aaaacgtgcg gctggaaatg gaacgtaacc atgagcgtta
tatgttcctg 480aaatggggaa agcaggcatt cagccgtttc agcgtggtgc
cgcccggcac cggcatttgc 540catcaggtta acctggaata cctgggtaaa
gccatctgga gcgaattaca ggacggggag 600tggattgctt acccggactc
gctggtgggg actgactccc atacgaccat gattaatggt 660ctgggcgtat
tggggtgggg cgtgggtggt attgaagcgg aagcggcgat gcttggtcag
720cccgtgtcga tgcttatccc ggatgtcgtc ggctttaagt taaccggtaa
acttcgggag 780gggatcactg ccactgacct ggtgctaacc gtcacgcaaa
tgctgcgtaa gcatggcgtc 840gtgggtaaat ttgttgaatt ttatggtgac
ggtctggatt cgctgccgtt ggcggatcgc 900gcgactatcg ctaatatgtc
gccggaatat ggcgccacct gtggtttctt ccccattgac 960gccatcacct
tggaatatat gcgattaagc ggacgtagcg acgatctgat cgagctggtt
1020gaaacctacg cgaaggcgca gggaatgtgg cgtaatcccg gtgacgaacc
ggtatttacc 1080agtacgctgg aactggatat gggcgatgtc gaggccagcc
tggccgggcc gaaacgcccg 1140caggatcgcg tggcgttagg cgatgtgccg
aaagcctttg ccgccagcgc cgagctggag 1200ctgaataccg cgcaaagaga
tcgccagccg gttgactata cgatgaacgg acagccatat 1260cagcttccgg
atggcgctgt cgtcattgcc gccatcacct cctgtacgaa tacctcgaat
1320cccagcgtgc tgatggcggc gggattactg gcgaaaaagg cagtaacgct
ggggttgaag 1380cgtcaaccgt gggtcaaggc ttctctggcg ccggggtcaa
aagtggtatc tgactatctg 1440gcgcaggcca aacttacgcc ttatctggat
gagctcggtt ttaacctggt cggctatggc 1500tgtacgacct gtatcgggaa
ctccggtccg ttgccggagc ctattgagac cgcgattaaa 1560aaaggcgatc
tgacggtagg ggccgtgctt tccggtaacc gaaattttga agggcgtatc
1620catccgctgg tgaaaacgaa ctggctggtg tcgccgccgc tggtggtcgc
gtatgcgctg 1680gccggaaaca tgaatattaa cctcgcgaca gacccgctgg
ggtacgatcg taaaggcgat 1740ccggtatacc tgaaggatat ctggccttcg
gcgcaggaaa ttgcccgcgc cgttgaactg 1800gtatcatcgg atatgttccg
taaagagtat gcggaagtgt ttgagggcac ggaagaatgg 1860aaatcgattc
aggttgaatc gtccgatacc tacggctggc agtcggattc aacctatatc
1920cgcctgtcgc ctttctttga tgaaatgcag gcccagcctg cacccgtcaa
agatatccac 1980ggcgcgcgta tcctggcgat gctgggcgat tcggtgacga
ccgaccatat ttccccggcc 2040ggcagtatca agccggacag tcccgccgga
cgctatctgc aaaaccacgg cgttgagcgg 2100aaggatttta actcctatgg
atcacggcgc ggcaaccatg aagtgatgat gcgcggtacg 2160ttcgccaata
ttcgtattcg caacgaaatg ctgcccggcg tcgaaggtgg gatgacgcgg
2220catttgccgg gtacggaagc gatgtcgatt tatgatgccg cgatgctcta
ccagcaggaa 2280aaaacgccgc tggcggtaat tgccgggaaa gagtatgggt
cgggatcgag ccgtgactgg 2340gcggcaaaag gtccgcggct gttaggtatt
cgcgtggtga tcgccgagtc gttcgaacgt 2400atccatcgct caagcctgat
tgggatgggg atcctgccgc tggagtttcc acagggcgta 2460acgcgtaaaa
cgctgggact gaccggggaa gaggtgattg atatcgcgga tctgcaaaat
2520ctgcgccctg gcgcgaccat tccggttatg ttaacgagag cggacggcag
caaagaaacg 2580gtgccttgtc gctgtcgcat tgataccgcc accgagctga
cttactacca gaatgacggc 2640atattgcact atgtcattag aaatatgctg aactaa
267628891PRTSalmonella enterica 28Met Ser Ser Thr Leu Arg Glu Ala
Ser Lys Asp Thr Leu Gln Ala Lys1 5 10 15Asp Lys Thr Tyr His Tyr Tyr
Ser Leu Pro Leu Ala Ala Lys Ser Leu 20 25 30Gly Asp Ile Ala Arg Leu
Pro Lys Ser Leu Lys Val Leu Leu Glu Asn 35 40 45Leu Leu Arg Trp Gln
Asp Gly Glu Ser Val Thr Asp Glu Asp Ile Gln 50 55 60Ala Leu Ala Gly
Trp Leu Lys Asn Ala His Ala Asp Arg Glu Ile Ala65 70 75 80Trp Arg
Pro Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala 85 90 95Val
Val Asp Leu Ala Ala Met Arg Glu Ala Val Lys Arg Leu Gly Gly 100 105
110Asp Thr Ser Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125His Ser
Val Thr Val Asp His Phe Gly Asp Asp Asp Ala Phe Glu Glu 130 135
140Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg Tyr Met Phe
Leu145 150 155 160Lys Trp Gly Lys Gln Ala Phe Ser Arg Phe Ser Val
Val Pro Pro Gly 165 170 175Thr Gly Ile Cys His Gln Val Asn Leu Glu
Tyr Leu Gly Lys Ala Ile 180 185 190Trp Ser Glu Leu Gln Asp Gly Glu
Trp Ile Ala Tyr Pro Asp Ser Leu 195 200 205Val Gly Thr Asp Ser His
Thr Thr Met Ile Asn Gly Leu Gly Val Leu 210 215 220Gly Trp Gly Val
Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln225 230 235 240Pro
Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr Gly 245 250
255Lys Leu Arg Glu Gly Ile Thr Ala Thr Asp Leu Val Leu Thr Val Thr
260 265 270Gln Met Leu Arg Lys His Gly Val Val Gly Lys Phe Val Glu
Phe Tyr 275 280 285Gly Asp Gly Leu Asp Ser Leu Pro Leu Ala Asp Arg
Ala Thr Ile Ala 290 295 300Asn Met Ser Pro Glu Tyr Gly Ala Thr Cys
Gly Phe Phe Pro Ile Asp305 310 315 320Ala Ile Thr Leu Glu Tyr Met
Arg Leu Ser Gly Arg Ser Asp Asp Leu 325 330 335Ile Glu Leu Val Glu
Thr Tyr Ala Lys Ala Gln Gly Met Trp Arg Asn 340 345 350Pro Gly Asp
Glu Pro Val Phe Thr Ser Thr Leu Glu Leu Asp Met Gly 355 360 365Asp
Val Glu Ala Ser Leu Ala Gly Pro Lys Arg Pro Gln Asp Arg Val 370 375
380Ala Leu Gly Asp Val Pro Lys Ala Phe Ala Ala Ser Ala Glu Leu
Glu385 390 395 400Leu Asn Thr Ala Gln Arg Asp Arg Gln Pro Val Asp
Tyr Thr Met Asn 405 410 415Gly Gln Pro Tyr Gln Leu Pro Asp Gly Ala
Val Val Ile Ala Ala Ile 420 425 430Thr Ser Cys Thr Asn Thr Ser Asn
Pro Ser Val Leu Met Ala Ala Gly 435 440 445Leu Leu Ala Lys Lys Ala
Val Thr Leu Gly Leu Lys Arg Gln Pro Trp 450 455 460Val Lys Ala Ser
Leu Ala Pro Gly Ser Lys Val Val Ser Asp Tyr Leu465 470 475 480Ala
Gln Ala Lys Leu Thr Pro Tyr Leu Asp Glu Leu Gly Phe Asn Leu 485 490
495Val Gly Tyr Gly Cys Thr Thr Cys Ile Gly Asn Ser Gly Pro Leu Pro
500 505 510Glu Pro Ile Glu Thr Ala Ile Lys Lys Gly Asp Leu Thr Val
Gly Ala 515 520 525Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Ile
His Pro Leu Val 530 535 540Lys Thr Asn Trp Leu Val Ser Pro Pro Leu
Val Val Ala Tyr Ala Leu545 550 555 560Ala Gly Asn Met Asn Ile Asn
Leu Ala Thr Asp Pro Leu Gly Tyr Asp 565 570 575Arg Lys Gly Asp Pro
Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln 580 585 590Glu Ile Ala
Arg Ala Val Glu Leu Val Ser Ser Asp Met Phe Arg Lys 595 600 605Glu
Tyr Ala Glu Val Phe Glu Gly Thr Glu Glu Trp Lys Ser Ile Gln 610 615
620Val Glu Ser Ser Asp Thr Tyr Gly Trp Gln Ser Asp Ser Thr Tyr
Ile625 630 635 640Arg Leu Ser Pro Phe Phe Asp Glu Met Gln Ala Gln
Pro Ala Pro Val 645 650 655Lys Asp Ile His Gly Ala Arg Ile Leu Ala
Met Leu Gly Asp Ser Val 660 665 670Thr Thr Asp His Ile Ser Pro Ala
Gly Ser Ile Lys Pro Asp Ser Pro 675 680 685Ala Gly Arg Tyr Leu Gln
Asn His Gly Val Glu Arg Lys Asp Phe Asn 690 695 700Ser Tyr Gly Ser
Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr705 710 715 720Phe
Ala Asn Ile Arg Ile Arg Asn Glu Met Leu Pro Gly Val Glu Gly 725 730
735Gly Met Thr Arg His Leu Pro Gly Thr Glu Ala Met Ser Ile Tyr Asp
740 745 750Ala Ala Met Leu Tyr Gln Gln Glu Lys Thr Pro Leu Ala Val
Ile Ala 755 760 765Gly Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp
Ala Ala Lys Gly 770 775 780Pro Arg Leu Leu Gly Ile Arg Val Val Ile
Ala Glu Ser Phe Glu Arg785 790 795 800Ile His Arg Ser Ser Leu Ile
Gly Met Gly Ile Leu Pro Leu Glu Phe 805 810 815Pro Gln Gly Val Thr
Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Val 820 825 830Ile Asp Ile
Ala Asp Leu Gln Asn Leu Arg Pro Gly Ala Thr Ile Pro 835 840 845Val
Met Leu Thr Arg Ala Asp Gly Ser Lys Glu Thr Val Pro Cys Arg 850 855
860Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr Gln Asn Asp
Gly865 870 875 880Ile Leu His Tyr Val Ile Arg Asn Met Leu Asn 885
890292598DNAEscherichia coli 29gtgctagaag aataccgtaa gcacgtagct
gagcgtgccg ctgaggggat tgcgcccaaa 60cccctggatg caaaccaaat ggccgcactt
gtagagctgc tgaaaaaccc gcccgcgggc 120gaagaagaat tcctgttaga
tctgttaacc aaccgtgttc ccccaggcgt cgatgaagcc 180gcctatgtca
aagcaggctt cctggctgct atcgcgaaag gcgaagccaa atcccctctg
240ctgactccgg aaaaagccat cgaactgctg ggcaccatgc agggtggtta
caacattcat 300ccgctgatcg acgcgctgga tgatgccaaa ctggcaccta
ttgctgccaa agcactttct 360cacacgctgc tgatgttcga taacttctat
gacgtagaag agaaagcgaa agcaggcaac 420gaatatgcga agcaggttat
gcagtcctgg gcggatgccg aatggttcct gaatcgcccg 480gcgctggctg
aaaaactgac cgttactgtc ttcaaagtca ctggcgaaac taacaccgat
540gacctttctc cggcaccgga tgcgtggtca cgcccggata tcccactgca
cgcgctggcg 600atgctgaaaa acgcccgtga aggtattgag ccagaccagc
ctggtgttgt tggtccgatc 660aagcaaatcg aagctctgca acagaaaggt
ttcccgctgg cgtacgtcgg tgacgttgtg 720ggtacgggtt cttcgcgtaa
atccgccact aactccgttc tgtggtttat gggcgatgat 780attccacatg
tgccgaacaa acgcggcggt ggtttgtgcc tcggcggtaa aattgcaccc
840atcttcttta acacgatgga agacgcgggt gcactgccaa tcgaagtcga
cgtctctaac 900ctgaacatgg gcgacgtgat tgacgtttac ccgtacaaag
gtgaagtgcg taaccacgaa 960accggcgaac tgctggcgac cttcgaactg
aaaaccgacg tgctgattga tgaagtgcgt 1020gctggtggcc gtattccgct
gattatcggg cgtggcctga ccaccaaagc gcgtgaagca 1080cttggtctgc
cgcacagtga tgtgttccgt caggcgaaag atgtcgctga gagcgatcgc
1140ggcttctcgc tggcgcaaaa aatggtaggc cgtgcctgtg gcgtgaaagg
cattcgtccg 1200ggcgcgtact gtgaaccgaa aatgacttct gtaggttccc
aggacaccac cggcccgatg 1260acccgtgatg aactgaaaga cctggcgtgc
ctgggcttct cggctgacct ggtgatgcag 1320tctttctgcc acaccgcggc
gtatccgaag ccagttgacg tgaacacgca ccacacgctg 1380ccggacttca
ttatgaaccg tggcggtgtg tcgctgcgtc cgggtgacgg cgtcattcac
1440tcctggctga accgtatgct gctgccggat accgtcggta ccggtggtga
ctcccatacc 1500cgtttcccga tcggtatctc tttcccggcg ggttctggtc
tggtggcgtt tgctgccgca 1560actggcgtaa tgccgcttga tatgccggaa
tccgttctgg tgcgcttcaa aggcaaaatg 1620cagccgggca tcaccctgcg
cgatctggta cacgctattc cgctgtatgc gatcaaacaa 1680ggtctgctga
ccgttgagaa gaaaggcaag aaaaacatct tctctggccg catcctggaa
1740attgaaggtc tgccggatct gaaagttgag caggcctttg agctaaccga
tgcgtccgcc 1800gagcgttctg ccgctggttg taccatcaag ctgaacaaag
aaccgatcat cgaatacctg 1860aactctaaca tcgtcctgct gaagtggatg
atcgcggaag gttacggcga tcgtcgtacc 1920ctggaacgtc gtattcaggg
catggaaaaa tggctggcga atcctgagct gctggaagcc 1980gatgcagatg
cggaatacgc ggcagtgatc gacatcgatc tggcggatat taaagagcca
2040atcctgtgtg ctccgaacga cccggatgac gcgcgtccgc tgtctgcggt
acagggtgag 2100aagatcgacg aagtgtttat cggttcctgc atgaccaaca
tcggtcactt ccgtgctgcg 2160ggtaaactgc tggatgcgca taaaggtcag
ttgccgaccc gcctgtgggt ggcaccgcca 2220acccgtatgg acgccgcaca
gttgaccgaa gaaggctact acagcgtctt cggtaagagt 2280ggtgcgcgta
tcgagatccc tggctgttcc ctgtgtatgg gtaaccaggc gcgtgtggcg
2340gacggtgcaa cggtggtttc cacctctacc cgtaacttcc cgaaccgtct
gggtactggc 2400gcgaatgtct tcctggcttc tgcggaactg gcggctgttg
cggcgctgat tggcaaactg 2460ccgacgccgg aagagtacca gacctacgtg
gcgcaggtag ataaaacagc cgttgatact 2520taccgttatc tgaacttcaa
ccagctttct cagtacaccg agaaagccga tggggtgatt 2580ttccagactg cggtttaa
259830865PRTEscherichia coli 30Met Leu Glu Glu Tyr Arg Lys His Val
Ala Glu Arg Ala Ala Glu Gly1 5 10 15Ile Ala Pro Lys Pro Leu Asp Ala
Asn Gln Met Ala Ala Leu Val Glu 20 25 30Leu Leu Lys Asn Pro Pro Ala
Gly Glu Glu Glu Phe Leu Leu Asp Leu 35 40 45Leu Thr Asn Arg Val Pro
Pro Gly Val Asp Glu Ala Ala Tyr Val Lys 50 55 60Ala Gly Phe Leu Ala
Ala Ile Ala Lys Gly Glu Ala Lys Ser Pro Leu65 70 75 80Leu Thr Pro
Glu Lys Ala Ile Glu Leu Leu Gly Thr Met Gln Gly Gly 85 90 95Tyr Asn
Ile His Pro Leu Ile Asp Ala Leu Asp Asp Ala Lys Leu Ala 100 105
110Pro Ile Ala Ala Lys Ala Leu Ser His Thr Leu Leu Met Phe Asp Asn
115 120 125Phe Tyr Asp Val Glu Glu Lys Ala Lys Ala Gly Asn Glu Tyr
Ala Lys 130 135 140Gln Val Met Gln Ser Trp Ala Asp Ala Glu Trp Phe
Leu Asn Arg Pro145 150 155 160Ala Leu Ala Glu Lys Leu Thr Val Thr
Val Phe Lys Val Thr Gly Glu 165 170 175Thr Asn Thr Asp Asp Leu Ser
Pro Ala Pro Asp Ala Trp Ser Arg Pro 180 185 190Asp Ile Pro Leu His
Ala Leu Ala Met Leu Lys Asn Ala Arg Glu Gly 195 200 205Ile Glu Pro
Asp Gln Pro Gly Val Val Gly Pro Ile Lys Gln Ile Glu 210 215 220Ala
Leu Gln Gln Lys Gly Phe Pro Leu Ala Tyr Val Gly Asp Val Val225 230
235 240Gly Thr Gly Ser Ser Arg Lys Ser Ala Thr Asn Ser Val Leu Trp
Phe 245 250 255Met Gly Asp Asp Ile Pro His Val Pro Asn Lys Arg Gly
Gly Gly Leu 260 265 270Cys Leu Gly Gly Lys Ile Ala Pro Ile Phe Phe
Asn Thr Met Glu Asp 275 280 285Ala Gly Ala Leu Pro Ile Glu Val Asp
Val Ser Asn Leu Asn Met Gly 290 295 300Asp Val Ile Asp Val Tyr Pro
Tyr Lys Gly Glu Val Arg Asn His Glu305 310 315 320Thr Gly Glu Leu
Leu Ala Thr Phe Glu Leu Lys Thr Asp Val Leu Ile 325 330 335Asp Glu
Val Arg Ala Gly Gly Arg Ile Pro Leu Ile Ile Gly Arg Gly 340 345
350Leu Thr Thr Lys Ala Arg Glu Ala Leu Gly Leu Pro His Ser Asp Val
355 360 365Phe Arg Gln Ala Lys Asp Val Ala Glu Ser Asp Arg Gly Phe
Ser Leu 370 375 380Ala Gln Lys Met Val Gly Arg Ala Cys Gly Val Lys
Gly Ile Arg Pro385 390 395 400Gly Ala Tyr Cys Glu Pro Lys Met Thr
Ser Val Gly Ser Gln Asp Thr 405 410 415Thr Gly Pro Met Thr Arg Asp
Glu Leu Lys Asp Leu Ala Cys Leu Gly 420 425 430Phe Ser Ala Asp Leu
Val Met Gln Ser Phe Cys His Thr Ala Ala Tyr 435 440 445Pro Lys Pro
Val Asp Val Asn Thr His His Thr Leu Pro Asp Phe Ile 450 455 460Met
Asn Arg Gly Gly Val Ser Leu Arg Pro Gly Asp Gly Val Ile His465 470
475 480Ser Trp Leu Asn Arg Met Leu Leu Pro Asp Thr Val Gly Thr Gly
Gly 485 490 495Asp Ser His Thr Arg Phe Pro Ile Gly Ile Ser Phe Pro
Ala Gly Ser 500 505 510Gly Leu Val Ala Phe Ala Ala Ala Thr Gly Val
Met Pro Leu Asp Met 515 520 525Pro Glu Ser Val Leu Val Arg Phe Lys
Gly Lys Met Gln Pro Gly Ile 530 535 540Thr Leu Arg Asp Leu Val His
Ala Ile Pro Leu Tyr Ala Ile Lys Gln545 550 555 560Gly Leu Leu Thr
Val Glu Lys Lys Gly Lys Lys Asn Ile Phe Ser Gly 565 570 575Arg Ile
Leu Glu Ile Glu Gly Leu Pro Asp Leu Lys Val Glu Gln Ala 580 585
590Phe Glu Leu Thr Asp Ala Ser Ala Glu Arg Ser Ala Ala Gly Cys Thr
595 600 605Ile Lys Leu Asn Lys Glu Pro Ile Ile Glu Tyr Leu Asn Ser
Asn Ile 610 615 620Val Leu Leu Lys Trp Met Ile Ala Glu Gly Tyr Gly
Asp Arg Arg Thr625 630 635 640Leu Glu Arg Arg Ile Gln Gly Met Glu
Lys Trp Leu Ala Asn Pro Glu 645 650 655Leu Leu Glu Ala Asp Ala Asp
Ala Glu Tyr Ala Ala Val Ile Asp Ile 660 665 670Asp Leu Ala Asp Ile
Lys Glu Pro Ile Leu Cys Ala Pro Asn Asp Pro 675 680 685Asp Asp Ala
Arg Pro Leu Ser Ala Val Gln Gly Glu Lys Ile Asp Glu 690 695 700Val
Phe Ile Gly Ser Cys Met Thr Asn Ile Gly His Phe Arg Ala Ala705 710
715 720Gly Lys Leu Leu Asp Ala His Lys Gly Gln Leu Pro Thr Arg Leu
Trp 725 730 735Val Ala Pro Pro Thr Arg Met Asp Ala Ala Gln Leu Thr
Glu Glu Gly 740 745 750Tyr Tyr Ser Val Phe Gly Lys Ser Gly Ala Arg
Ile Glu Ile Pro Gly 755 760 765Cys Ser Leu Cys Met Gly Asn Gln Ala
Arg Val Ala Asp Gly Ala Thr 770 775 780Val Val Ser Thr Ser Thr Arg
Asn Phe Pro Asn Arg Leu Gly Thr Gly785 790 795 800Ala Asn Val Phe
Leu Ala Ser Ala Glu Leu Ala Ala Val Ala Ala Leu 805 810 815Ile Gly
Lys Leu Pro Thr Pro Glu Glu Tyr Gln Thr Tyr Val Ala Gln 820 825
830Val Asp Lys Thr Ala Val Asp Thr Tyr Arg Tyr Leu Asn Phe Asn Gln
835 840 845Leu Ser Gln Tyr Thr Glu Lys Ala Asp Gly Val Ile Phe Gln
Thr Ala 850 855 860Val865312697DNAPantoea ananatis 31atgctgcccc
acgcggaacc gggcacctta ccctactcat ggaatgcgtt gtcatcaggg 60tgcaccggaa
acccatacaa tgagagcgag gagaacgtcg tgctagaaga ataccgtaag
120cacgttgccg agcgtgctgc ccaagggatc gtacctaagc cattagatgc
ttcccaaatg 180gccgcgctgg ttgaactgct aaaaaatcca cctgcgggtg
aagaagaatt tttgaccgat 240ttgttggtca accgcgtacc acccggcgtc
gatgaagcgg cgtatgttaa agcaggtttc 300ctggctgctg tcgccaaagg
cgaaacaacc tctcctctgg tatctcctga aaaagctgtt 360gaactgctcg
gtaccatgca gggcggctac aacatccatc ctctgattga agcattagat
420gatgcgaaac tcgcaccgat tgcggcaaaa gcgctttccc acacgttgct
gatgtttgac 480agcttttacg acgttgaaga aaaagccaaa gcaggcaatc
cacacgcgaa gcaggtgatg 540cagtcgtggg cggatgccga atggtatctg
tcacgtcctg agctggctga aaaaattacc 600gttacggttt tcaaagtcac
gggtgaaacc aacaccgatg acctgtctcc ggcaccggat 660gcctggtcac
gtccggatat cccactgcat gccctggcga tgctgaaaaa tgcccgtgaa
720ggcattgagc cgaatgaggc aggcaacatc ggtccgatta agcagatcga
agcactgcag 780gccaaaggtt tcccgctggc ctatgtgggc gacgttgtgg
gcacaggttc atcccgtaaa 840tcggccacca actcggtgct gtggtttatg
ggcgatgaca tccctaacgt gccgaataag 900aaaggcggtg gtgttgttct
gggcggcaag attgcgccta tcttcttcaa caccatggaa 960gatgccggcg
cgctgccgat cgaagtggat gtgaacgacc tgaatatggg tgatgtcatt
1020gatatctatc cttttaaagg cgaagtgcgc aatcacgaaa ccggcgatct
gctggccagt 1080tttgaactga aaaccgacgt gctgattgat gaagtgcgtg
ccggtggccg tattccactg 1140atcatcggtc gtggcctgac cagcaaagcg
cgtgaatccc tggggctgcc tgtcagcacc 1200gtgttccgta tcgcgaaaga
cgtggcggaa agctcacgcg gctactcact ggcgcagaaa 1260atggttggcc
gcgcctgtgg cgtggacggc gtccgtccgg gtgcctattg cgaaccgaaa
1320atgacctcgg tgggctcgca ggataccacc ggtcctatga cccgcgacga
gctgaaagac 1380ctggcatgtc tgggcttctc tgccgatctg gtgatgcagt
cgttctgtca cacggcggct 1440tatcctaagc cagtcgatgt gaacacgcac
cacacgctgc cagacttcat catgaaccgt 1500ggcggcgttt ccctgcgtcc
gggggatggc gtgattcact cctggctgaa ccgtatgctg 1560ctgccggata
ccgtcggcac cggtggcgat tcccacaccc gcttcccaat cggtatttcc
1620ttcccggcag gttcgggtct ggttgccttt gcggcagcga ccggcgttat
gccactggat 1680atgccggaat cggtactggt acgtttcaaa ggtaaaatgc
agcccggcat tacgctgcgc 1740gatctggttc acgccattcc gctgtatgcg
attaagcagg gcctgttaac cgttgagaag 1800aaaggtaaga agaacatctt
ctccggtcgc atccttgaaa ttgaaggtct gcccgatctg 1860aaagtggagc
aggcgttcga actgaccgat gcctctgctg agcgttctgc tgcaggctgt
1920accatcaagc tggatcaggc gccgatcaaa gagtatctga catctaacat
tgtcctgctg 1980aaatggatga tctcagaagg ctacggcgat cgtcgtacgc
ttgagcgccg tatcgaaggc 2040atggagaaat ggctggcaga tccacagctg
ctcgaagccg acgccgatgc agaatatgct 2100gcggtgattg acatcgatct
ggcggagatc aaagagccga ttctgtgcgc gccaaacgat 2160cccgacgatg
cgcgtctgct gtcagatgtg cagggcgaga agattgatga agtctttatc
2220ggttcgtgca tgaccaacat cggtcacttc cgcgcggcag gtaagttgct
ggacagccac 2280aaaggccaac tgccaacccg tttatgggtc gcaccaccga
ccaaaatgga tgcggcacag 2340ctgacggaag aaggctacta cagcgtgttc
ggtaagagcg gtgcgcgtat tgagatcccg 2400ggttgttcac tgtgcatggg
taaccaggca cgtgtcgccg atggcgcaac ggtcgtttcg
2460acctcaaccc gtaacttccc gaaccgtttg ggtacagggg ctaacgtcta
cctggcttct 2520gcggagctgg cggcggtctc atcactgtta ggtaagctgc
caacgccaga tgagtatcag 2580cagtttatgg cgcaggtgga taaaacggct
agcgatacct atcgctatct caactttgac 2640cagttgagcc agtacactga
aaaagcggat ggcgtgattt tccagaccgc cgtctga 269732898PRTPantoea
ananatis 32Met Leu Pro His Ala Glu Pro Gly Thr Leu Pro Tyr Ser Trp
Asn Ala1 5 10 15Leu Ser Ser Gly Cys Thr Gly Asn Pro Tyr Asn Glu Ser
Glu Glu Asn 20 25 30Val Val Leu Glu Glu Tyr Arg Lys His Val Ala Glu
Arg Ala Ala Gln 35 40 45Gly Ile Val Pro Lys Pro Leu Asp Ala Ser Gln
Met Ala Ala Leu Val 50 55 60Glu Leu Leu Lys Asn Pro Pro Ala Gly Glu
Glu Glu Phe Leu Thr Asp65 70 75 80Leu Leu Val Asn Arg Val Pro Pro
Gly Val Asp Glu Ala Ala Tyr Val 85 90 95Lys Ala Gly Phe Leu Ala Ala
Val Ala Lys Gly Glu Thr Thr Ser Pro 100 105 110Leu Val Ser Pro Glu
Lys Ala Val Glu Leu Leu Gly Thr Met Gln Gly 115 120 125Gly Tyr Asn
Ile His Pro Leu Ile Glu Ala Leu Asp Asp Ala Lys Leu 130 135 140Ala
Pro Ile Ala Ala Lys Ala Leu Ser His Thr Leu Leu Met Phe Asp145 150
155 160Ser Phe Tyr Asp Val Glu Glu Lys Ala Lys Ala Gly Asn Pro His
Ala 165 170 175Lys Gln Val Met Gln Ser Trp Ala Asp Ala Glu Trp Tyr
Leu Ser Arg 180 185 190Pro Glu Leu Ala Glu Lys Ile Thr Val Thr Val
Phe Lys Val Thr Gly 195 200 205Glu Thr Asn Thr Asp Asp Leu Ser Pro
Ala Pro Asp Ala Trp Ser Arg 210 215 220Pro Asp Ile Pro Leu His Ala
Leu Ala Met Leu Lys Asn Ala Arg Glu225 230 235 240Gly Ile Glu Pro
Asn Glu Ala Gly Asn Ile Gly Pro Ile Lys Gln Ile 245 250 255Glu Ala
Leu Gln Ala Lys Gly Phe Pro Leu Ala Tyr Val Gly Asp Val 260 265
270Val Gly Thr Gly Ser Ser Arg Lys Ser Ala Thr Asn Ser Val Leu Trp
275 280 285Phe Met Gly Asp Asp Ile Pro Asn Val Pro Asn Lys Lys Gly
Gly Gly 290 295 300Val Val Leu Gly Gly Lys Ile Ala Pro Ile Phe Phe
Asn Thr Met Glu305 310 315 320Asp Ala Gly Ala Leu Pro Ile Glu Val
Asp Val Asn Asp Leu Asn Met 325 330 335Gly Asp Val Ile Asp Ile Tyr
Pro Phe Lys Gly Glu Val Arg Asn His 340 345 350Glu Thr Gly Asp Leu
Leu Ala Ser Phe Glu Leu Lys Thr Asp Val Leu 355 360 365Ile Asp Glu
Val Arg Ala Gly Gly Arg Ile Pro Leu Ile Ile Gly Arg 370 375 380Gly
Leu Thr Ser Lys Ala Arg Glu Ser Leu Gly Leu Pro Val Ser Thr385 390
395 400Val Phe Arg Ile Ala Lys Asp Val Ala Glu Ser Ser Arg Gly Tyr
Ser 405 410 415Leu Ala Gln Lys Met Val Gly Arg Ala Cys Gly Val Asp
Gly Val Arg 420 425 430Pro Gly Ala Tyr Cys Glu Pro Lys Met Thr Ser
Val Gly Ser Gln Asp 435 440 445Thr Thr Gly Pro Met Thr Arg Asp Glu
Leu Lys Asp Leu Ala Cys Leu 450 455 460Gly Phe Ser Ala Asp Leu Val
Met Gln Ser Phe Cys His Thr Ala Ala465 470 475 480Tyr Pro Lys Pro
Val Asp Val Asn Thr His His Thr Leu Pro Asp Phe 485 490 495Ile Met
Asn Arg Gly Gly Val Ser Leu Arg Pro Gly Asp Gly Val Ile 500 505
510His Ser Trp Leu Asn Arg Met Leu Leu Pro Asp Thr Val Gly Thr Gly
515 520 525Gly Asp Ser His Thr Arg Phe Pro Ile Gly Ile Ser Phe Pro
Ala Gly 530 535 540Ser Gly Leu Val Ala Phe Ala Ala Ala Thr Gly Val
Met Pro Leu Asp545 550 555 560Met Pro Glu Ser Val Leu Val Arg Phe
Lys Gly Lys Met Gln Pro Gly 565 570 575Ile Thr Leu Arg Asp Leu Val
His Ala Ile Pro Leu Tyr Ala Ile Lys 580 585 590Gln Gly Leu Leu Thr
Val Glu Lys Lys Gly Lys Lys Asn Ile Phe Ser 595 600 605Gly Arg Ile
Leu Glu Ile Glu Gly Leu Pro Asp Leu Lys Val Glu Gln 610 615 620Ala
Phe Glu Leu Thr Asp Ala Ser Ala Glu Arg Ser Ala Ala Gly Cys625 630
635 640Thr Ile Lys Leu Asp Gln Ala Pro Ile Lys Glu Tyr Leu Thr Ser
Asn 645 650 655Ile Val Leu Leu Lys Trp Met Ile Ser Glu Gly Tyr Gly
Asp Arg Arg 660 665 670Thr Leu Glu Arg Arg Ile Glu Gly Met Glu Lys
Trp Leu Ala Asp Pro 675 680 685Gln Leu Leu Glu Ala Asp Ala Asp Ala
Glu Tyr Ala Ala Val Ile Asp 690 695 700Ile Asp Leu Ala Glu Ile Lys
Glu Pro Ile Leu Cys Ala Pro Asn Asp705 710 715 720Pro Asp Asp Ala
Arg Leu Leu Ser Asp Val Gln Gly Glu Lys Ile Asp 725 730 735Glu Val
Phe Ile Gly Ser Cys Met Thr Asn Ile Gly His Phe Arg Ala 740 745
750Ala Gly Lys Leu Leu Asp Ser His Lys Gly Gln Leu Pro Thr Arg Leu
755 760 765Trp Val Ala Pro Pro Thr Lys Met Asp Ala Ala Gln Leu Thr
Glu Glu 770 775 780Gly Tyr Tyr Ser Val Phe Gly Lys Ser Gly Ala Arg
Ile Glu Ile Pro785 790 795 800Gly Cys Ser Leu Cys Met Gly Asn Gln
Ala Arg Val Ala Asp Gly Ala 805 810 815Thr Val Val Ser Thr Ser Thr
Arg Asn Phe Pro Asn Arg Leu Gly Thr 820 825 830Gly Ala Asn Val Tyr
Leu Ala Ser Ala Glu Leu Ala Ala Val Ser Ser 835 840 845Leu Leu Gly
Lys Leu Pro Thr Pro Asp Glu Tyr Gln Gln Phe Met Ala 850 855 860Gln
Val Asp Lys Thr Ala Ser Asp Thr Tyr Arg Tyr Leu Asn Phe Asp865 870
875 880Gln Leu Ser Gln Tyr Thr Glu Lys Ala Asp Gly Val Ile Phe Gln
Thr 885 890 895Ala Val332598DNAPectobacterium atrosepticum
33gtgctagaag aatatcgtaa gcacgtagcc gagcgggctg cgcaggggat tgttcctaaa
60ccgttagatg ccacgcagat ggccgcgctg gttgagtcac tcaagaatcc cccggcaggc
120gaagaggaag tattgcttga tctgctgatt aaccgcgttc cacccggtgt
tgatgaagcc 180gcctacgtga aggccggttt tttggctgct gtcgctaaag
gcgaagccac ttcccccttg 240gttacccagg aaaaagcgat tgagctgctg
ggcaccatgc aaggtggtta taatattcat 300ccgctaattg atgcattaga
cagcgacacg ctggcaccga ttgccgcgaa agcgttgtcc 360cagacgctgc
tgatgtttga taacttctat gatgtggaag aaaaagcgaa agcaggcaat
420gcacacgcca agaaagtgat tcaatcctgg gctgatgccg agtggttcct
gtcccgcccg 480aaactggcgg aaaaaattac cgttaccgtc tttaaagtca
ctggtgaaac taacactgat 540gacctgtctc cggcacctga tgcctggtcg
cgccctgata tcccgctgca cgcgttggcg 600atgctgaaaa atgcccgtga
aggcattgat cccgatcagc ctggcaacgt gggtccgatc 660aaacagatcg
aagaactgaa caagaaaggc ttcccgctgg cgtacgtcgg tgacgtcgtt
720ggtacgggat cgtcgcgtaa atctgcaacc aactccgtgc tgtggttcat
gggtgaagac 780attcctcatg ttccgaacaa acgcggcggt ggtgtggtgc
tgggcggcaa gattgcccca 840atcttcttca acaccatgga agatgccggt
gcgttgccga tcgaagtcga tgttaacgat 900ctgaatatgg gcgatgtgat
cgacatctac ccgtatgaag gtgaagttcg ccgtcatgac 960acgaatgaag
tgctggcgac gtttgcgctg aagaccgacg tattactgga tgaagtgcgc
1020gccggtggcc gtattccgtt gatcatcgga cgtgggttga cgtctaaagc
gcgtgagtca 1080ctggacttgc cgcacagcga tgtcttccgt atttctaaag
ccattgaagc cagcaaaaaa 1140ggcttctcac tggcgcagaa aatggtcggt
cgcgcctgcg gcgtcgcggg tattcgccct 1200gatgaatact gcgaacctaa
gatgacatcc gtgggttcac aggacaccac cgggccgatg 1260actcgtgatg
agctgaaaga tctggcttgt ctcggcttct ccgctgactt ggtgatgcag
1320tcgttctgtc acactgcggc ctatcctaag ccggttgacg tgaccacgca
ccacacgctg 1380cctgatttca tcatgaaccg tggcggcgta tcgctacgtc
cgggcgatgg cgttatccac 1440tcctggctga accgtatgct gctgccggat
accgttggta caggcggtga ctcccacacc 1500cgtttcccaa tcggtatctc
tttccctgca ggttctgggc tggtggcgtt tgctgcggct 1560acgggcgtga
tgccgctgga tatgccggaa tctattctgg tgcgcttcaa aggcaaaatg
1620cagccgggta ttactctgcg cgatctggtt cacgccattc cgctgtatgc
catcaaacaa 1680ggtctgctga ccgttgagaa gaaaggtaag aagaacatct
tctctggtcg tattctggaa 1740atcgaaggcc tgccggatct gaaagtcgag
caggcgtttg aactgaccga cgcctcggcg 1800gaacgttctg cggctggttg
tacgatcaag ctggataaag cgccgatcat cgagtatttg 1860aactccaaca
tcgttctgct gaagtggatg atctccgaag gttatggcga tcgccgtacg
1920ctggaacgtc gtgttcaagg tatggaaaaa tggctggccg atccgcaact
gctggaagcc 1980gatgccgatg ccgagtatgc ggcggtgatc gacatcgatc
tggctgacat caaagagccg 2040atcctgtgtg cgccgaacga tccagacgat
gcgcgctggc tgtctgacgt gcagggcgag 2100aagattgatg aagtcttcat
cggttcctgt atgaccaaca tcggacactt ccgtgcggcg 2160ggtaaactgc
tggatagcca caaaggccag ctgccgaccc gtttgtgggt tgcgccgccg
2220accaaaatgg acgcggcaca gctgacggaa gaaggctatt acagcgtgtt
tggtaagagc 2280ggagcgcgta tcgagatccc aggttgctcg ctgtgcatgg
gtaatcaggc gcgcgtggcg 2340gatggcgcga cggtcgtttc tacgtcgacc
cgtaacttcc cgaaccgttt gggaaccggt 2400gctaacgtgt atcttgcgtc
tgctgaactg gctgcggttg cctcgctgtt gggccgtttg 2460ccgacgcctg
aagagtacca gacctacatg tcgcaggtgg ataaaaccgc gcaggacacc
2520tatcgctatc tgaattttga ccagttaggt caatatactg agaaagccga
tggcgtgatc 2580ttccagacga ctgtctaa 259834865PRTPectobacterium
atrosepticum 34Met Leu Glu Glu Tyr Arg Lys His Val Ala Glu Arg Ala
Ala Gln Gly1 5 10 15Ile Val Pro Lys Pro Leu Asp Ala Thr Gln Met Ala
Ala Leu Val Glu 20 25 30Ser Leu Lys Asn Pro Pro Ala Gly Glu Glu Glu
Val Leu Leu Asp Leu 35 40 45Leu Ile Asn Arg Val Pro Pro Gly Val Asp
Glu Ala Ala Tyr Val Lys 50 55 60Ala Gly Phe Leu Ala Ala Val Ala Lys
Gly Glu Ala Thr Ser Pro Leu65 70 75 80Val Thr Gln Glu Lys Ala Ile
Glu Leu Leu Gly Thr Met Gln Gly Gly 85 90 95Tyr Asn Ile His Pro Leu
Ile Asp Ala Leu Asp Ser Asp Thr Leu Ala 100 105 110Pro Ile Ala Ala
Lys Ala Leu Ser Gln Thr Leu Leu Met Phe Asp Asn 115 120 125Phe Tyr
Asp Val Glu Glu Lys Ala Lys Ala Gly Asn Ala His Ala Lys 130 135
140Lys Val Ile Gln Ser Trp Ala Asp Ala Glu Trp Phe Leu Ser Arg
Pro145 150 155 160Lys Leu Ala Glu Lys Ile Thr Val Thr Val Phe Lys
Val Thr Gly Glu 165 170 175Thr Asn Thr Asp Asp Leu Ser Pro Ala Pro
Asp Ala Trp Ser Arg Pro 180 185 190Asp Ile Pro Leu His Ala Leu Ala
Met Leu Lys Asn Ala Arg Glu Gly 195 200 205Ile Asp Pro Asp Gln Pro
Gly Asn Val Gly Pro Ile Lys Gln Ile Glu 210 215 220Glu Leu Asn Lys
Lys Gly Phe Pro Leu Ala Tyr Val Gly Asp Val Val225 230 235 240Gly
Thr Gly Ser Ser Arg Lys Ser Ala Thr Asn Ser Val Leu Trp Phe 245 250
255Met Gly Glu Asp Ile Pro His Val Pro Asn Lys Arg Gly Gly Gly Val
260 265 270Val Leu Gly Gly Lys Ile Ala Pro Ile Phe Phe Asn Thr Met
Glu Asp 275 280 285Ala Gly Ala Leu Pro Ile Glu Val Asp Val Asn Asp
Leu Asn Met Gly 290 295 300Asp Val Ile Asp Ile Tyr Pro Tyr Glu Gly
Glu Val Arg Arg His Asp305 310 315 320Thr Asn Glu Val Leu Ala Thr
Phe Ala Leu Lys Thr Asp Val Leu Leu 325 330 335Asp Glu Val Arg Ala
Gly Gly Arg Ile Pro Leu Ile Ile Gly Arg Gly 340 345 350Leu Thr Ser
Lys Ala Arg Glu Ser Leu Asp Leu Pro His Ser Asp Val 355 360 365Phe
Arg Ile Ser Lys Ala Ile Glu Ala Ser Lys Lys Gly Phe Ser Leu 370 375
380Ala Gln Lys Met Val Gly Arg Ala Cys Gly Val Ala Gly Ile Arg
Pro385 390 395 400Asp Glu Tyr Cys Glu Pro Lys Met Thr Ser Val Gly
Ser Gln Asp Thr 405 410 415Thr Gly Pro Met Thr Arg Asp Glu Leu Lys
Asp Leu Ala Cys Leu Gly 420 425 430Phe Ser Ala Asp Leu Val Met Gln
Ser Phe Cys His Thr Ala Ala Tyr 435 440 445Pro Lys Pro Val Asp Val
Thr Thr His His Thr Leu Pro Asp Phe Ile 450 455 460Met Asn Arg Gly
Gly Val Ser Leu Arg Pro Gly Asp Gly Val Ile His465 470 475 480Ser
Trp Leu Asn Arg Met Leu Leu Pro Asp Thr Val Gly Thr Gly Gly 485 490
495Asp Ser His Thr Arg Phe Pro Ile Gly Ile Ser Phe Pro Ala Gly Ser
500 505 510Gly Leu Val Ala Phe Ala Ala Ala Thr Gly Val Met Pro Leu
Asp Met 515 520 525Pro Glu Ser Ile Leu Val Arg Phe Lys Gly Lys Met
Gln Pro Gly Ile 530 535 540Thr Leu Arg Asp Leu Val His Ala Ile Pro
Leu Tyr Ala Ile Lys Gln545 550 555 560Gly Leu Leu Thr Val Glu Lys
Lys Gly Lys Lys Asn Ile Phe Ser Gly 565 570 575Arg Ile Leu Glu Ile
Glu Gly Leu Pro Asp Leu Lys Val Glu Gln Ala 580 585 590Phe Glu Leu
Thr Asp Ala Ser Ala Glu Arg Ser Ala Ala Gly Cys Thr 595 600 605Ile
Lys Leu Asp Lys Ala Pro Ile Ile Glu Tyr Leu Asn Ser Asn Ile 610 615
620Val Leu Leu Lys Trp Met Ile Ser Glu Gly Tyr Gly Asp Arg Arg
Thr625 630 635 640Leu Glu Arg Arg Val Gln Gly Met Glu Lys Trp Leu
Ala Asp Pro Gln 645 650 655Leu Leu Glu Ala Asp Ala Asp Ala Glu Tyr
Ala Ala Val Ile Asp Ile 660 665 670Asp Leu Ala Asp Ile Lys Glu Pro
Ile Leu Cys Ala Pro Asn Asp Pro 675 680 685Asp Asp Ala Arg Trp Leu
Ser Asp Val Gln Gly Glu Lys Ile Asp Glu 690 695 700Val Phe Ile Gly
Ser Cys Met Thr Asn Ile Gly His Phe Arg Ala Ala705 710 715 720Gly
Lys Leu Leu Asp Ser His Lys Gly Gln Leu Pro Thr Arg Leu Trp 725 730
735Val Ala Pro Pro Thr Lys Met Asp Ala Ala Gln Leu Thr Glu Glu Gly
740 745 750Tyr Tyr Ser Val Phe Gly Lys Ser Gly Ala Arg Ile Glu Ile
Pro Gly 755 760 765Cys Ser Leu Cys Met Gly Asn Gln Ala Arg Val Ala
Asp Gly Ala Thr 770 775 780Val Val Ser Thr Ser Thr Arg Asn Phe Pro
Asn Arg Leu Gly Thr Gly785 790 795 800Ala Asn Val Tyr Leu Ala Ser
Ala Glu Leu Ala Ala Val Ala Ser Leu 805 810 815Leu Gly Arg Leu Pro
Thr Pro Glu Glu Tyr Gln Thr Tyr Met Ser Gln 820 825 830Val Asp Lys
Thr Ala Gln Asp Thr Tyr Arg Tyr Leu Asn Phe Asp Gln 835 840 845Leu
Gly Gln Tyr Thr Glu Lys Ala Asp Gly Val Ile Phe Gln Thr Thr 850 855
860Val865352598DNASalmonella enterica 35gtgctagaag aataccgtaa
gcacgtagct gagcgtgctg cccaggggat tgtgccgaaa 60cctttagacg caacccaaat
ggctgcgctt gtcgagctgc tgaagacccc gcctgtgggc 120gaagaagaat
tcctgttaga cctgttgatc aaccgcgttc ctcctggcgt cgatgaagcc
180gcttatgtta aagccggttt tctcgctgct gtcgcgaaag gcgacaccac
ctccccgctg 240gtctccccag aaaaagccat tgaactgctg ggcaccatgc
agggtggtta caacattcat 300ccgctgattg acgcgctgga cgatgcgaaa
ctggcgccga ttgcggccaa agcgctgtct 360cacaccctgc tgatgttcga
taacttctac gacgtagaag agaaagccaa agcgggcaat 420gaatatgcca
aacaggtgat gcaatcttgg gccgacgccg aatggttcct gagccgtccg
480ccgctggcgg aaaaaatcac cgtcaccgtt ttcaaagtga ccggcgaaac
gaataccgac 540gatctctctc cggcgccgga tgcgtggtcg agaccggata
tcccgttaca tgcgcaggcg 600atgctgaaaa acgcccgtga aggcattgag
ccggatcagc caggcgttgt cggcccgatc 660aaacaaatcg aagcattgca
gaaaaaaggc tacccgctgg cctacgtggg tgacgtggtg 720ggcaccggtt
cttcccgtaa atccgcgacc aactccgtgc tgtggttcat gggcgatgac
780atcccgaacg tgccgaacaa gcgcggcggc ggtctgtgcc tcggcggcaa
aattgcgcct 840atcttcttta acaccatgga agatgcgggc gcgctgccga
ttgaagttga cgtttctaac 900ctgaacatgg gcgatgtaat tgacgtctac
ccgtacaaag gcgaagtgcg caatcatgaa 960accgatgaac tgctggcaac
cttcgaactg aaaaccgacg tgctgatcga cgaagtacgc 1020gccggtggcc
gtattccgct gattatcgga cgtggcctga ccaccaaagc gcgtgaagcg
1080ctgggtctgc cgcactctga cgttttccgt caggcaaaag acgtggcaga
aagcagccgt 1140ggcttctctc tggcgcagaa aatggtcggt cgcgcctgcg
gcgtgaaagg cattcgtccg 1200ggcgcgtact gcgaaccgaa aatgacctcc
gtcggttctc aggatactac tggcccgatg 1260acccgtgatg agctgaaaga
cctggcctgt ctgggattct ccgccgatct ggtcatgcag 1320tcgttctgtc
acaccgcagc ctatccgaag cccgttgacg tcaccacgca ccacacgctg
1380ccggacttca ttatgaaccg cggcggtgtc tccctgcgtc cgggcgacgg
cgtgatccac 1440tcctggctga accgtatgct gctgccggac accgtcggta
ccggcggtga ctcccatacc 1500cgtttcccga ttggtatctc tttcccggcg
ggttctggtc tggtggcgtt tgccgccgcg 1560accggcgtga tgccgctgga
tatgccggaa tcggtgctgg tgcgcttcaa aggcaaaatg 1620cagccgggca
tcaccctgcg cgatctggtc catgccatcc cgctgtacgc catcaaacag
1680ggcctgctga ccgttgagaa gaaaggcaag aaaaacatct tctctggccg
catcctggaa 1740atcgaaggtc tgccggatct gaaagtcgag caggcgtttg
agctgaccga tgcttctgcc 1800gagcgttccg ctgccggttg taccatcaag
ctgaacaaag agccgatcgt tgaatacctg 1860acctccaaca tcgtcctgct
gaagtggatg atcgccgaag gctacggcga ccgtcgtacg 1920ctggaacgtc
gtatccaggg tatggaaaaa tggctggcgg acccgcagct gctggaagcc
1980gatgctgacg cggaatacgc agcggtgatc gacatcgatc tggcggatat
caaagagcca 2040atcctctgtg cgccgaacga tccggacgac gcgcgtctgc
tgtctgacgt gcagggcgag 2100aagatcgacg aagtgttcat cggttcctgc
atgaccaaca tcggccactt ccgcgcggct 2160ggtaagctgc tggatagcca
caaaggccag ttgccaaccc gcctgtgggt agcgccgcca 2220acccgtatgg
acgctgcgca gctgaccgaa gaaggttact acagcgtgtt tggtaagagc
2280ggtgcgcgta tcgaaatccc gggttgttcc ctgtgtatgg gtaaccaggc
gcgtgtggct 2340gacggcgcga cggtggtttc cacttctacc cgtaacttcc
cgaaccgttt aggtactggt 2400gcgaacgtct tcctggcttc tgcggagctg
gcggcggttg cagcgcttat tggcaaactg 2460ccgacgccgg aagagtacca
gacctttgtg gcgcaggtgg ataagacggc ggtggatacc 2520taccgttatc
tgaacttcga ccagctctct cagtacactg agaaagcgga tggggtgatt
2580ttccagactg cggtataa 259836865PRTSalmonella enterica 36Met Leu
Glu Glu Tyr Arg Lys His Val Ala Glu Arg Ala Ala Gln Gly1 5 10 15Ile
Val Pro Lys Pro Leu Asp Ala Thr Gln Met Ala Ala Leu Val Glu 20 25
30Leu Leu Lys Thr Pro Pro Val Gly Glu Glu Glu Phe Leu Leu Asp Leu
35 40 45Leu Ile Asn Arg Val Pro Pro Gly Val Asp Glu Ala Ala Tyr Val
Lys 50 55 60Ala Gly Phe Leu Ala Ala Val Ala Lys Gly Asp Thr Thr Ser
Pro Leu65 70 75 80Val Ser Pro Glu Lys Ala Ile Glu Leu Leu Gly Thr
Met Gln Gly Gly 85 90 95Tyr Asn Ile His Pro Leu Ile Asp Ala Leu Asp
Asp Ala Lys Leu Ala 100 105 110Pro Ile Ala Ala Lys Ala Leu Ser His
Thr Leu Leu Met Phe Asp Asn 115 120 125Phe Tyr Asp Val Glu Glu Lys
Ala Lys Ala Gly Asn Glu Tyr Ala Lys 130 135 140Gln Val Met Gln Ser
Trp Ala Asp Ala Glu Trp Phe Leu Ser Arg Pro145 150 155 160Pro Leu
Ala Glu Lys Ile Thr Val Thr Val Phe Lys Val Thr Gly Glu 165 170
175Thr Asn Thr Asp Asp Leu Ser Pro Ala Pro Asp Ala Trp Ser Arg Pro
180 185 190Asp Ile Pro Leu His Ala Gln Ala Met Leu Lys Asn Ala Arg
Glu Gly 195 200 205Ile Glu Pro Asp Gln Pro Gly Val Val Gly Pro Ile
Lys Gln Ile Glu 210 215 220Ala Leu Gln Lys Lys Gly Tyr Pro Leu Ala
Tyr Val Gly Asp Val Val225 230 235 240Gly Thr Gly Ser Ser Arg Lys
Ser Ala Thr Asn Ser Val Leu Trp Phe 245 250 255Met Gly Asp Asp Ile
Pro Asn Val Pro Asn Lys Arg Gly Gly Gly Leu 260 265 270Cys Leu Gly
Gly Lys Ile Ala Pro Ile Phe Phe Asn Thr Met Glu Asp 275 280 285Ala
Gly Ala Leu Pro Ile Glu Val Asp Val Ser Asn Leu Asn Met Gly 290 295
300Asp Val Ile Asp Val Tyr Pro Tyr Lys Gly Glu Val Arg Asn His
Glu305 310 315 320Thr Asp Glu Leu Leu Ala Thr Phe Glu Leu Lys Thr
Asp Val Leu Ile 325 330 335Asp Glu Val Arg Ala Gly Gly Arg Ile Pro
Leu Ile Ile Gly Arg Gly 340 345 350Leu Thr Thr Lys Ala Arg Glu Ala
Leu Gly Leu Pro His Ser Asp Val 355 360 365Phe Arg Gln Ala Lys Asp
Val Ala Glu Ser Ser Arg Gly Phe Ser Leu 370 375 380Ala Gln Lys Met
Val Gly Arg Ala Cys Gly Val Lys Gly Ile Arg Pro385 390 395 400Gly
Ala Tyr Cys Glu Pro Lys Met Thr Ser Val Gly Ser Gln Asp Thr 405 410
415Thr Gly Pro Met Thr Arg Asp Glu Leu Lys Asp Leu Ala Cys Leu Gly
420 425 430Phe Ser Ala Asp Leu Val Met Gln Ser Phe Cys His Thr Ala
Ala Tyr 435 440 445Pro Lys Pro Val Asp Val Thr Thr His His Thr Leu
Pro Asp Phe Ile 450 455 460Met Asn Arg Gly Gly Val Ser Leu Arg Pro
Gly Asp Gly Val Ile His465 470 475 480Ser Trp Leu Asn Arg Met Leu
Leu Pro Asp Thr Val Gly Thr Gly Gly 485 490 495Asp Ser His Thr Arg
Phe Pro Ile Gly Ile Ser Phe Pro Ala Gly Ser 500 505 510Gly Leu Val
Ala Phe Ala Ala Ala Thr Gly Val Met Pro Leu Asp Met 515 520 525Pro
Glu Ser Val Leu Val Arg Phe Lys Gly Lys Met Gln Pro Gly Ile 530 535
540Thr Leu Arg Asp Leu Val His Ala Ile Pro Leu Tyr Ala Ile Lys
Gln545 550 555 560Gly Leu Leu Thr Val Glu Lys Lys Gly Lys Lys Asn
Ile Phe Ser Gly 565 570 575Arg Ile Leu Glu Ile Glu Gly Leu Pro Asp
Leu Lys Val Glu Gln Ala 580 585 590Phe Glu Leu Thr Asp Ala Ser Ala
Glu Arg Ser Ala Ala Gly Cys Thr 595 600 605Ile Lys Leu Asn Lys Glu
Pro Ile Val Glu Tyr Leu Thr Ser Asn Ile 610 615 620Val Leu Leu Lys
Trp Met Ile Ala Glu Gly Tyr Gly Asp Arg Arg Thr625 630 635 640Leu
Glu Arg Arg Ile Gln Gly Met Glu Lys Trp Leu Ala Asp Pro Gln 645 650
655Leu Leu Glu Ala Asp Ala Asp Ala Glu Tyr Ala Ala Val Ile Asp Ile
660 665 670Asp Leu Ala Asp Ile Lys Glu Pro Ile Leu Cys Ala Pro Asn
Asp Pro 675 680 685Asp Asp Ala Arg Leu Leu Ser Asp Val Gln Gly Glu
Lys Ile Asp Glu 690 695 700Val Phe Ile Gly Ser Cys Met Thr Asn Ile
Gly His Phe Arg Ala Ala705 710 715 720Gly Lys Leu Leu Asp Ser His
Lys Gly Gln Leu Pro Thr Arg Leu Trp 725 730 735Val Ala Pro Pro Thr
Arg Met Asp Ala Ala Gln Leu Thr Glu Glu Gly 740 745 750Tyr Tyr Ser
Val Phe Gly Lys Ser Gly Ala Arg Ile Glu Ile Pro Gly 755 760 765Cys
Ser Leu Cys Met Gly Asn Gln Ala Arg Val Ala Asp Gly Ala Thr 770 775
780Val Val Ser Thr Ser Thr Arg Asn Phe Pro Asn Arg Leu Gly Thr
Gly785 790 795 800Ala Asn Val Phe Leu Ala Ser Ala Glu Leu Ala Ala
Val Ala Ala Leu 805 810 815Ile Gly Lys Leu Pro Thr Pro Glu Glu Tyr
Gln Thr Phe Val Ala Gln 820 825 830Val Asp Lys Thr Ala Val Asp Thr
Tyr Arg Tyr Leu Asn Phe Asp Gln 835 840 845Leu Ser Gln Tyr Thr Glu
Lys Ala Asp Gly Val Ile Phe Gln Thr Ala 850 855
860Val865371539DNAEscherichia coli 37atgaccaata atcccccttc
agcacagatt aagcccggcg agtatggttt ccccctcaag 60ttaaaagccc gctatgacaa
ctttattggc ggcgaatggg tagcccctgc cgacggcgag 120tattaccaga
atctgacgcc ggtgaccggg cagctgctgt gcgaagtggc gtcttcgggc
180aaacgagaca tcgatctggc gctggatgct gcgcacaaag tgaaagataa
atgggcgcac 240acctcggtgc aggatcgtgc ggcgattctg tttaagattg
ccgatcgaat ggaacaaaac 300ctcgagctgt tagcgacagc tgaaacctgg
gataacggca aacccattcg cgaaaccagt 360gctgcggatg taccgctggc
gattgaccat ttccgctatt tcgcctcgtg tattcgggcg 420caggaaggtg
ggatcagtga agttgatagc gaaaccgtgg cctatcattt ccatgaaccg
480ttaggcgtgg tggggcagat tatcccgtgg aacttcccgc tgctgatggc
gagctggaaa 540atggctcccg cgctggcggc gggcaactgt gtggtgctga
aacccgcacg tcttaccccg 600ctttctgtac tgctgctaat ggaaattgtc
ggtgatttac tgccgccggg cgtggtgaac 660gtggtcaatg gcgcaggtgg
ggtaattggc gaatatctgg cgacctcgaa acgcatcgcc 720aaagtggcgt
ttaccggctc aacggaagtg ggccaacaaa ttatgcaata cgcaacgcaa
780aacattattc cggtgacgct ggagttgggc ggtaagtcgc caaatatctt
ctttgctgat 840gtgatggatg aagaagatgc ctttttcgat aaagcgctgg
aaggctttgc actgtttgcc 900tttaaccagg gcgaagtttg cacctgtccg
agtcgtgctt tagtgcagga atctatctac 960gaacgcttta tggaacgcgc
catccgccgt gtcgaaagca ttcgtagcgg taacccgctc 1020gacagcgtga
cgcaaatggg cgcgcaggtt tctcacgggc aactggaaac catcctcaac
1080tacattgata tcggtaaaaa agagggcgct gacgtgctca caggcgggcg
gcgcaagctg 1140ctggaaggtg aactgaaaga cggctactac ctcgaaccga
cgattctgtt tggtcagaac 1200aatatgcggg tgttccagga ggagattttt
ggcccggtgc tggcggtgac caccttcaaa 1260acgatggaag aagcgctgga
gctggcgaac gatacgcaat atggcctggg cgcgggcgtc 1320tggagccgca
acggtaatct ggcctataag atggggcgcg gcatacaggc tgggcgcgtg
1380tggaccaact gttatcacgc ttacccggca catgcggcgt ttggtggcta
caaacaatca 1440ggtatcggtc gcgaaaccca caagatgatg ctggagcatt
accagcaaac caagtgcctg 1500ctggtgagct actcggataa accgttgggg
ctgttctga 153938512PRTEscherichia coli 38Met Thr Asn Asn Pro Pro
Ser Ala Gln Ile Lys Pro Gly Glu Tyr Gly1 5 10 15Phe Pro Leu Lys Leu
Lys Ala Arg Tyr Asp Asn Phe Ile Gly Gly Glu 20 25 30Trp Val Ala Pro
Ala Asp Gly Glu Tyr Tyr Gln Asn Leu Thr Pro Val 35 40 45Thr Gly Gln
Leu Leu Cys Glu Val Ala Ser Ser Gly Lys Arg Asp Ile 50 55 60Asp Leu
Ala Leu Asp Ala Ala His Lys Val Lys Asp Lys Trp Ala His65 70 75
80Thr Ser Val Gln Asp Arg Ala Ala Ile Leu Phe Lys Ile Ala Asp Arg
85 90 95Met Glu Gln Asn Leu Glu Leu Leu Ala Thr Ala Glu Thr Trp Asp
Asn 100 105 110Gly Lys Pro Ile Arg Glu Thr Ser Ala Ala Asp Val Pro
Leu Ala Ile 115 120 125Asp His Phe Arg Tyr Phe Ala Ser Cys Ile Arg
Ala Gln Glu Gly Gly 130 135 140Ile Ser Glu Val Asp Ser Glu Thr Val
Ala Tyr His Phe His Glu Pro145 150 155 160Leu Gly Val Val Gly Gln
Ile Ile Pro Trp Asn Phe Pro Leu Leu Met 165 170 175Ala Ser Trp Lys
Met Ala Pro Ala Leu Ala Ala Gly Asn Cys Val Val 180 185 190Leu Lys
Pro Ala Arg Leu Thr Pro Leu Ser Val Leu Leu Leu Met Glu 195 200
205Ile Val Gly Asp Leu Leu Pro Pro Gly Val Val Asn Val Val Asn Gly
210 215 220Ala Gly Gly Val Ile Gly Glu Tyr Leu Ala Thr Ser Lys Arg
Ile Ala225 230 235 240Lys Val Ala Phe Thr Gly Ser Thr Glu Val Gly
Gln Gln Ile Met Gln 245 250 255Tyr Ala Thr Gln Asn Ile Ile Pro Val
Thr Leu Glu Leu Gly Gly Lys 260 265 270Ser Pro Asn Ile Phe Phe Ala
Asp Val Met Asp Glu Glu Asp Ala Phe 275 280 285Phe Asp Lys Ala Leu
Glu Gly Phe Ala Leu Phe Ala Phe Asn Gln Gly 290 295 300Glu Val Cys
Thr Cys Pro Ser Arg Ala Leu Val Gln Glu Ser Ile Tyr305 310 315
320Glu Arg Phe Met Glu Arg Ala Ile Arg Arg Val Glu Ser Ile Arg Ser
325 330 335Gly Asn Pro Leu Asp Ser Val Thr Gln Met Gly Ala Gln Val
Ser His 340 345 350Gly Gln Leu Glu Thr Ile Leu Asn Tyr Ile Asp Ile
Gly Lys Lys Glu 355 360 365Gly Ala Asp Val Leu Thr Gly Gly Arg Arg
Lys Leu Leu Glu Gly Glu 370 375 380Leu Lys Asp Gly Tyr Tyr Leu Glu
Pro Thr Ile Leu Phe Gly Gln Asn385 390 395 400Asn Met Arg Val Phe
Gln Glu Glu Ile Phe Gly Pro Val Leu Ala Val 405 410 415Thr Thr Phe
Lys Thr Met Glu Glu Ala Leu Glu Leu Ala Asn Asp Thr 420 425 430Gln
Tyr Gly Leu Gly Ala Gly Val Trp Ser Arg Asn Gly Asn Leu Ala 435 440
445Tyr Lys Met Gly Arg Gly Ile Gln Ala Gly Arg Val Trp Thr Asn Cys
450 455 460Tyr His Ala Tyr Pro Ala His Ala Ala Phe Gly Gly Tyr Lys
Gln Ser465 470 475 480Gly Ile Gly Arg Glu Thr His Lys Met Met Leu
Glu His Tyr Gln Gln 485 490 495Thr Lys Cys Leu Leu Val Ser Tyr Ser
Asp Lys Pro Leu Gly Leu Phe 500 505 510391473DNAPantoea ananatis
39atgtcagatt ttgatcccga taaggtccgt ctttcgaccg gacattttat caacggccag
60tttgtttcgg catccggcaa aacaatgggg attaaacgtc cctcggatgg ccagcattat
120gccgacatca acgaagcggg cgcagaaacg gtcggcgagg cggtaagcct
cgcggaagac 180gcgcgcattc gcagcggctg gtcaagctgc tcgccacgcg
aacggggcgc ggccatttcc 240cgctgggcgg atttgatcga tgccgacaaa
gattatctgg cgcagcttga ggcggtgggc 300tccacgcgtc cgataacgga
cacgatcaat attgaagtgc ctttcaccgc cgctgctctg 360cgtttttatg
cagaatgcgc agacaagtac agcggagatg tgtttccaac ccagaacagt
420agcctgggga tgctggtgcc ggagccttat ggcgtgatcg gcgcgatcac
gccgtggaat 480ttcccgctgt cgatggcgtc gtggaagtgc ggcccggcgc
tggctgcggg aaacgcggtg 540gtactcaagc cctctgagct gacgccattc
tccaccgcgc ggctggctga gctggccgtg 600caggccggta ttccgcccgg
tgtgctcaac gtcgttcagg gcggcggaca ggtcaccggc 660aatgcactgg
tgacccatcc tctggtcaga aaggtgtcct ttaccggttc aaccgcgacc
720ggggcggcga tcatgagcca ggccgcgttg cacggtacca agcccgtgac
gctggaactg 780ggcggaaaaa gcccgcagct ggtttttgac gatgcaggcg
atgcggatga aatcgccgag 840cgcctgtttc tgggcttcac cgtcaacgcc
ggtcaggcct gtgtttcagg gacgcgtctg 900atcattcaac aaggtatcgc
agaacgggtc atcgaaaagc ttatcgccct atgcaaaaca 960ccgctgcccg
gtatgacctg gcaggccgcc acgcgttact gtccgctgat cgatcaacgt
1020cagggggaaa aagtcgcgac gattatcgcg cagtccgtgg cgcagggggc
cagcattctg 1080gccggcggcc agcgttacga aaacaccgcg cagggctggt
tctggcaacc gacgctgtta 1140gccaatgtga atcaggataa cgtcgcgatt
caggaggaga tcttcggacc ggtgctcacc 1200atccagacgt ttagcgatga
agaagaagcc ctggcgctcg cacagcatcg cgtttttggc 1260ttatgtgcag
gcgtgcatac gctcaacatg ccacgtgcga tgcggctgat gaaagccctg
1320gacagcggta cggtctggat caaccgctat cgtcgaacct gggactttat
cattccaacc 1380ggcggctttc aggggtccgg ttttggcaaa gatcttgggc
gccaggcctt tgagtcatgt 1440cagcgctaca agagtgtctt aatcgatttt taa
147340490PRTPantoea ananatis 40Met Ser Asp Phe Asp Pro Asp Lys Val
Arg Leu Ser Thr Gly His Phe1 5 10 15Ile Asn Gly Gln Phe Val Ser Ala
Ser Gly Lys Thr Met Gly Ile Lys 20 25 30Arg Pro Ser Asp Gly Gln His
Tyr Ala Asp Ile Asn Glu Ala Gly Ala 35 40 45Glu Thr Val Gly Glu Ala
Val Ser Leu Ala Glu Asp Ala Arg Ile Arg 50 55 60Ser Gly Trp Ser Ser
Cys Ser Pro Arg Glu Arg Gly Ala Ala Ile Ser65 70 75 80Arg Trp Ala
Asp Leu Ile Asp Ala Asp Lys Asp Tyr Leu Ala Gln Leu 85 90 95Glu Ala
Val Gly Ser Thr Arg Pro Ile Thr Asp Thr Ile Asn Ile Glu 100 105
110Val Pro Phe Thr Ala Ala Ala Leu Arg Phe Tyr Ala Glu Cys Ala Asp
115 120 125Lys Tyr Ser Gly Asp Val Phe Pro Thr Gln Asn Ser Ser Leu
Gly Met 130 135 140Leu Val Pro Glu Pro Tyr Gly Val Ile Gly Ala Ile
Thr Pro Trp Asn145 150 155 160Phe Pro Leu Ser Met Ala Ser Trp Lys
Cys Gly Pro Ala Leu Ala Ala 165 170 175Gly Asn Ala Val Val Leu Lys
Pro Ser Glu Leu Thr Pro Phe Ser Thr 180 185 190Ala Arg Leu Ala Glu
Leu Ala Val Gln Ala Gly Ile Pro Pro Gly Val 195 200 205Leu Asn Val
Val Gln Gly Gly Gly Gln Val Thr Gly Asn Ala Leu Val 210 215 220Thr
His Pro Leu Val Arg Lys Val Ser Phe Thr Gly Ser Thr Ala Thr225 230
235 240Gly Ala Ala Ile Met Ser Gln Ala Ala Leu His Gly Thr Lys Pro
Val 245 250 255Thr Leu Glu Leu Gly Gly Lys Ser Pro Gln Leu Val Phe
Asp Asp Ala 260 265 270Gly Asp Ala Asp Glu Ile Ala Glu Arg Leu Phe
Leu Gly Phe Thr Val 275 280 285Asn Ala Gly Gln Ala Cys Val Ser Gly
Thr Arg Leu Ile Ile Gln Gln 290 295 300Gly Ile Ala Glu Arg Val Ile
Glu Lys Leu Ile Ala Leu Cys Lys Thr305 310 315 320Pro Leu Pro Gly
Met Thr Trp Gln Ala Ala Thr Arg Tyr Cys Pro Leu 325 330 335Ile Asp
Gln Arg Gln Gly Glu Lys Val Ala Thr Ile Ile Ala Gln Ser 340 345
350Val Ala Gln Gly Ala Ser Ile Leu Ala Gly Gly Gln Arg Tyr
Glu Asn 355 360 365Thr Ala Gln Gly Trp Phe Trp Gln Pro Thr Leu Leu
Ala Asn Val Asn 370 375 380Gln Asp Asn Val Ala Ile Gln Glu Glu Ile
Phe Gly Pro Val Leu Thr385 390 395 400Ile Gln Thr Phe Ser Asp Glu
Glu Glu Ala Leu Ala Leu Ala Gln His 405 410 415Arg Val Phe Gly Leu
Cys Ala Gly Val His Thr Leu Asn Met Pro Arg 420 425 430Ala Met Arg
Leu Met Lys Ala Leu Asp Ser Gly Thr Val Trp Ile Asn 435 440 445Arg
Tyr Arg Arg Thr Trp Asp Phe Ile Ile Pro Thr Gly Gly Phe Gln 450 455
460Gly Ser Gly Phe Gly Lys Asp Leu Gly Arg Gln Ala Phe Glu Ser
Cys465 470 475 480Gln Arg Tyr Lys Ser Val Leu Ile Asp Phe 485
490411536DNAPectobacterium atrosepticum 41atggcgcacg ataatctcga
aggccgatcc gcatttggcg aagtcggttc tctcgatctg 60aaaaaacgct atgacaattt
tatcggtgga acctgggttc cgcctgatgc tggtcagtat 120tttgtcaatt
taacgccagt gacaggccag ccgatgtgtg aagtagccag ttcgtcaacg
180cgagatattg accacgcgct ggatgcagcc cacaaggcaa aagcggaatg
gggtggtcta 240tcggtacagg aacgggcgct ggtgcttaac cgtattgccg
accggatgga acaaaatctt 300gaacggctag cgcaggtgga aacctgggat
aacggtaagc cgatacgtga aacaagcggg 360gcggatgtgc cgctggcgat
tgaccacttt cgttattttg ctgcctgtat ccgcgcgcaa 420gagggggcaa
tcagcgaaat tgacggcgat accgtggcct atcattttca tgagcctctt
480ggcgttgttg cgcaaatcat tccctggaac ttcccgctgc tgatggcctg
ttggaagatg 540gctcctgcac tggcagccgg taactgtatt gtactgaagc
ctgccaagct gacgccgatg 600tcggtgctga ttttgatgga gcttattcag
gatctgctgc ctgcgggggt cattaatgtc 660gtcaatggat cgggaagtga
gattggcgag tatctggcaa catcgaaacg cgttgcgaaa 720gtcgcattca
ccgggtcaac cgaagtgggc cagcagatca tgagctatgc ggcacagaat
780gtgacgccgg tgacgctgga attgggtggc aaatcgccga acatcttctt
tgccgatgtg 840atggataagg aagatagttt ctttgacaaa gcgctcgaag
gtttcacgct gtttgccttc 900aatcagggag aggtttgcac ctgcccgagc
cgcgcgctgg tgcaggaatc tatctatgat 960cgctttatgg aacgggcaat
caagcgcgtt gaagctatcc gcatcggtaa cccgctggac 1020agcaaaacca
tgatgggcgc acaggtgtca gcaggccagc tcgaaaccat ccttaactat
1080attgatatcg gtaagaaaga gggcgcacgg gtactcactg gcggccagcg
taaggcgatg 1140ccgggcgggc tggcggaagg ctactatttg gagccgacga
tattgttcgg taaaaatagt 1200atgcgtgtct ttcaggagga aattttcggt
ccggtgttgg cggtaacaac gttcaagacg 1260atggaagatg cgctggagat
agctaacgat acggaatacg gtctgggtgc tggcgtgtgg 1320agccgtaacg
gtaacgtcgc ttaccgaatg gggcgcggca ttcaggctgg tcgagtttgg
1380accaactgtt atcacgccta tccggcacat gccgcgtttg ggggctataa
gcagtccggt 1440atcgggcgtg agaatcataa aatgatgctg gatcattatc
agcaaaccaa gtgcctgttg 1500gtgagttact ctgataagcc gatggggctg ttctaa
153642511PRTPectobacterium atrosepticum 42Met Ala His Asp Asn Leu
Glu Gly Arg Ser Ala Phe Gly Glu Val Gly1 5 10 15Ser Leu Asp Leu Lys
Lys Arg Tyr Asp Asn Phe Ile Gly Gly Thr Trp 20 25 30Val Pro Pro Asp
Ala Gly Gln Tyr Phe Val Asn Leu Thr Pro Val Thr 35 40 45Gly Gln Pro
Met Cys Glu Val Ala Ser Ser Ser Thr Arg Asp Ile Asp 50 55 60His Ala
Leu Asp Ala Ala His Lys Ala Lys Ala Glu Trp Gly Gly Leu65 70 75
80Ser Val Gln Glu Arg Ala Leu Val Leu Asn Arg Ile Ala Asp Arg Met
85 90 95Glu Gln Asn Leu Glu Arg Leu Ala Gln Val Glu Thr Trp Asp Asn
Gly 100 105 110Lys Pro Ile Arg Glu Thr Ser Gly Ala Asp Val Pro Leu
Ala Ile Asp 115 120 125His Phe Arg Tyr Phe Ala Ala Cys Ile Arg Ala
Gln Glu Gly Ala Ile 130 135 140Ser Glu Ile Asp Gly Asp Thr Val Ala
Tyr His Phe His Glu Pro Leu145 150 155 160Gly Val Val Ala Gln Ile
Ile Pro Trp Asn Phe Pro Leu Leu Met Ala 165 170 175Cys Trp Lys Met
Ala Pro Ala Leu Ala Ala Gly Asn Cys Ile Val Leu 180 185 190Lys Pro
Ala Lys Leu Thr Pro Met Ser Val Leu Ile Leu Met Glu Leu 195 200
205Ile Gln Asp Leu Leu Pro Ala Gly Val Ile Asn Val Val Asn Gly Ser
210 215 220Gly Ser Glu Ile Gly Glu Tyr Leu Ala Thr Ser Lys Arg Val
Ala Lys225 230 235 240Val Ala Phe Thr Gly Ser Thr Glu Val Gly Gln
Gln Ile Met Ser Tyr 245 250 255Ala Ala Gln Asn Val Thr Pro Val Thr
Leu Glu Leu Gly Gly Lys Ser 260 265 270Pro Asn Ile Phe Phe Ala Asp
Val Met Asp Lys Glu Asp Ser Phe Phe 275 280 285Asp Lys Ala Leu Glu
Gly Phe Thr Leu Phe Ala Phe Asn Gln Gly Glu 290 295 300Val Cys Thr
Cys Pro Ser Arg Ala Leu Val Gln Glu Ser Ile Tyr Asp305 310 315
320Arg Phe Met Glu Arg Ala Ile Lys Arg Val Glu Ala Ile Arg Ile Gly
325 330 335Asn Pro Leu Asp Ser Lys Thr Met Met Gly Ala Gln Val Ser
Ala Gly 340 345 350Gln Leu Glu Thr Ile Leu Asn Tyr Ile Asp Ile Gly
Lys Lys Glu Gly 355 360 365Ala Arg Val Leu Thr Gly Gly Gln Arg Lys
Ala Met Pro Gly Gly Leu 370 375 380Ala Glu Gly Tyr Tyr Leu Glu Pro
Thr Ile Leu Phe Gly Lys Asn Ser385 390 395 400Met Arg Val Phe Gln
Glu Glu Ile Phe Gly Pro Val Leu Ala Val Thr 405 410 415Thr Phe Lys
Thr Met Glu Asp Ala Leu Glu Ile Ala Asn Asp Thr Glu 420 425 430Tyr
Gly Leu Gly Ala Gly Val Trp Ser Arg Asn Gly Asn Val Ala Tyr 435 440
445Arg Met Gly Arg Gly Ile Gln Ala Gly Arg Val Trp Thr Asn Cys Tyr
450 455 460His Ala Tyr Pro Ala His Ala Ala Phe Gly Gly Tyr Lys Gln
Ser Gly465 470 475 480Ile Gly Arg Glu Asn His Lys Met Met Leu Asp
His Tyr Gln Gln Thr 485 490 495Lys Cys Leu Leu Val Ser Tyr Ser Asp
Lys Pro Met Gly Leu Phe 500 505 510431539DNASalmonella enterica
43atgacgaaca atcccccttc aacacgtatt cagccaagtg aatacgggta cccactgaag
60ttaaaagccc gctatgacaa ttttattggc ggtgactggg ttgcgcccgc cgacggcgaa
120tattatcaaa acctgacgcc agtgaccggc cagccgctat gtgaagtcgc
ttcctccggt 180aaaaaagata tcgatttagc gctcgacgcc gcgcataagg
cgaaagataa gtgggcgcat 240acgtcagtac aagaccgtgc cgctatcttg
tttaagatcg ccgatcggat ggaacaaaac 300ctcgaactgt tggcgacagc
ggaaacttgg gataacggta aaccgattcg tgaaaccagt 360gccgccgaca
taccgctggc gatcgatcat ttccgctatt tcgcctcctg tatacgtgcg
420caggagggcg ggatcagcga agttgatagc gaaaccgtgg cctaccattt
tcacgaaccg 480cttggtgtcg tggggcagat aatcccgtgg aactttccgc
tgctgatggc aagctggaaa 540atggcgccag cgctggcggc aggtaactgc
gtggtgctta aaccggcacg cctgacgccg 600ctttccgttt tactgttaat
ggaagtcatt ggcgatctgt taccgccggg cgttgtcaac 660gtcgtgaacg
gcgcgggcgg cgagattggc gaatatctgg cgacctcaaa acgtatcgcg
720aaggtggcgt ttaccggttc gacggaagtg ggtcaacaga tcatgcagta
cgccacgcag 780aacattattc cggtgacgct ggagttaggc ggcaagtcgc
ccaatatctt cttcgccgac 840gtgatggatg aggaagatgc gttctttgat
aaagcgctgg agggatttgc cctgtttgcc 900tttaaccagg gcgaggtgtg
tacctgtcca agccgtgcgc tggttcagga gtccatctat 960gagcgcttta
tggagcgcgc tattcgccgg gtggagagca ttcgcagcgg gaacccgcta
1020gatagcggta cacagatggg agcgcaggtc tctcacggcc agcttgagac
tatcctcaat 1080tatatcgata tcggtaaaaa agagggggcc gatattctga
ccggtgggcg acgcaaggaa 1140ctggatggcg aacttaaaga gggctattac
cttgagccta ccattctgtt tggtaagaat 1200aatatgcgcg tctttcagga
ggagatcttt ggcccggtgc tggcggtaac cacctttaaa 1260accatggaga
aggcgctgga aatcgctaac gatacgcaat atggcctggg tgctggcgtc
1320tggagccgca acggcaatct ggcctataag atggggcgcg gcattcaggc
cgggcgcgta 1380tggaccaact gctatcacgc ctatccggca catgcggcgt
ttggcggcta taagcagtcg 1440ggcatcgggc gcgaaaccca taagatgatg
ctggaacact accagcaaac caagtgcctg 1500ttggtgagtt attccgataa
gccgctgggg ctgttctga 153944512PRTSalmonella enterica 44Met Thr Asn
Asn Pro Pro Ser Thr Arg Ile Gln Pro Ser Glu Tyr Gly1 5 10 15Tyr Pro
Leu Lys Leu Lys Ala Arg Tyr Asp Asn Phe Ile Gly Gly Asp 20 25 30Trp
Val Ala Pro Ala Asp Gly Glu Tyr Tyr Gln Asn Leu Thr Pro Val 35 40
45Thr Gly Gln Pro Leu Cys Glu Val Ala Ser Ser Gly Lys Lys Asp Ile
50 55 60Asp Leu Ala Leu Asp Ala Ala His Lys Ala Lys Asp Lys Trp Ala
His65 70 75 80Thr Ser Val Gln Asp Arg Ala Ala Ile Leu Phe Lys Ile
Ala Asp Arg 85 90 95Met Glu Gln Asn Leu Glu Leu Leu Ala Thr Ala Glu
Thr Trp Asp Asn 100 105 110Gly Lys Pro Ile Arg Glu Thr Ser Ala Ala
Asp Ile Pro Leu Ala Ile 115 120 125Asp His Phe Arg Tyr Phe Ala Ser
Cys Ile Arg Ala Gln Glu Gly Gly 130 135 140Ile Ser Glu Val Asp Ser
Glu Thr Val Ala Tyr His Phe His Glu Pro145 150 155 160Leu Gly Val
Val Gly Gln Ile Ile Pro Trp Asn Phe Pro Leu Leu Met 165 170 175Ala
Ser Trp Lys Met Ala Pro Ala Leu Ala Ala Gly Asn Cys Val Val 180 185
190Leu Lys Pro Ala Arg Leu Thr Pro Leu Ser Val Leu Leu Leu Met Glu
195 200 205Val Ile Gly Asp Leu Leu Pro Pro Gly Val Val Asn Val Val
Asn Gly 210 215 220Ala Gly Gly Glu Ile Gly Glu Tyr Leu Ala Thr Ser
Lys Arg Ile Ala225 230 235 240Lys Val Ala Phe Thr Gly Ser Thr Glu
Val Gly Gln Gln Ile Met Gln 245 250 255Tyr Ala Thr Gln Asn Ile Ile
Pro Val Thr Leu Glu Leu Gly Gly Lys 260 265 270Ser Pro Asn Ile Phe
Phe Ala Asp Val Met Asp Glu Glu Asp Ala Phe 275 280 285Phe Asp Lys
Ala Leu Glu Gly Phe Ala Leu Phe Ala Phe Asn Gln Gly 290 295 300Glu
Val Cys Thr Cys Pro Ser Arg Ala Leu Val Gln Glu Ser Ile Tyr305 310
315 320Glu Arg Phe Met Glu Arg Ala Ile Arg Arg Val Glu Ser Ile Arg
Ser 325 330 335Gly Asn Pro Leu Asp Ser Gly Thr Gln Met Gly Ala Gln
Val Ser His 340 345 350Gly Gln Leu Glu Thr Ile Leu Asn Tyr Ile Asp
Ile Gly Lys Lys Glu 355 360 365Gly Ala Asp Ile Leu Thr Gly Gly Arg
Arg Lys Glu Leu Asp Gly Glu 370 375 380Leu Lys Glu Gly Tyr Tyr Leu
Glu Pro Thr Ile Leu Phe Gly Lys Asn385 390 395 400Asn Met Arg Val
Phe Gln Glu Glu Ile Phe Gly Pro Val Leu Ala Val 405 410 415Thr Thr
Phe Lys Thr Met Glu Lys Ala Leu Glu Ile Ala Asn Asp Thr 420 425
430Gln Tyr Gly Leu Gly Ala Gly Val Trp Ser Arg Asn Gly Asn Leu Ala
435 440 445Tyr Lys Met Gly Arg Gly Ile Gln Ala Gly Arg Val Trp Thr
Asn Cys 450 455 460Tyr His Ala Tyr Pro Ala His Ala Ala Phe Gly Gly
Tyr Lys Gln Ser465 470 475 480Gly Ile Gly Arg Glu Thr His Lys Met
Met Leu Glu His Tyr Gln Gln 485 490 495Thr Lys Cys Leu Leu Val Ser
Tyr Ser Asp Lys Pro Leu Gly Leu Phe 500 505 510452676DNAEscherichia
coli 45atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgtgtaaa
aaaagcccag 60cgtgaatatg ccagtttcac tcaagagcaa gtagacaaaa tcttccgcgc
cgccgctctg 120gctgctgcag atgctcgaat cccactcgcg aaaatggccg
ttgccgaatc cggcatgggt 180atcgtcgaag ataaagtgat caaaaaccac
tttgcttctg aatatatcta caacgcctat 240aaagatgaaa aaacctgtgg
tgttctgtct gaagacgaca cttttggtac catcactatc 300gctgaaccaa
tcggtattat ttgcggtatc gttccgacca ctaacccgac ttcaactgct
360atcttcaaat cgctgatcag tctgaagacc cgtaacgcca ttatcttctc
cccgcacccg 420cgtgcaaaag atgccaccaa caaagcggct gatatcgttc
tgcaggctgc tatcgctgcc 480ggtgctccga aagatctgat cggctggatc
gatcaacctt ctgttgaact gtctaacgca 540ctgatgcacc acccagacat
caacctgatc ctcgcgactg gtggtccggg catggttaaa 600gccgcataca
gctccggtaa accagctatc ggtgtaggcg cgggcaacac tccagttgtt
660atcgatgaaa ctgctgatat caaacgtgca gttgcatctg tactgatgtc
caaaaccttc 720gacaacggcg taatctgtgc ttctgaacag tctgttgttg
ttgttgactc tgtttatgac 780gctgtacgtg aacgttttgc aacccacggc
ggctatctgt tgcagggtaa agagctgaaa 840gctgttcagg atgttatcct
gaaaaacggt gcgctgaacg cggctatcgt tggtcagcca 900gcctataaaa
ttgctgaact ggcaggcttc tctgtaccag aaaacaccaa gattctgatc
960ggtgaagtga ccgttgttga tgaaagcgaa ccgttcgcac atgaaaaact
gtccccgact 1020ctggcaatgt accgcgctaa agatttcgaa gacgcggtag
aaaaagcaga gaaactggtt 1080gctatgggcg gtatcggtca tacctcttgc
ctgtacactg accaggataa ccaaccggct 1140cgcgtttctt acttcggtca
gaaaatgaaa acggcgcgta tcctgattaa caccccagcg 1200tctcagggtg
gtatcggtga cctgtataac ttcaaactcg caccttccct gactctgggt
1260tgtggttctt ggggtggtaa ctccatctct gaaaacgttg gtccgaaaca
cctgatcaac 1320aagaaaaccg ttgctaagcg agctgaaaac atgttgtggc
acaaacttcc gaaatctatc 1380tacttccgcc gtggctccct gccaatcgcg
ctggatgaag tgattactga tggccacaaa 1440cgtgcgctca tcgtgactga
ccgcttcctg ttcaacaatg gttatgctga tcagatcact 1500tccgtactga
aagcagcagg cgttgaaact gaagtcttct tcgaagtaga agcggacccg
1560accctgagca tcgttcgtaa aggtgcagaa ctggcaaact ccttcaaacc
agacgtgatt 1620atcgcgctgg gtggtggttc cccgatggac gccgcgaaga
tcatgtgggt tatgtacgaa 1680catccggaaa ctcacttcga agagctggcg
ctgcgcttta tggatatccg taaacgtatc 1740tacaagttcc cgaaaatggg
cgtgaaagcg aaaatgatcg ctgtcaccac cacttctggt 1800acaggttctg
aagtcactcc gtttgcggtt gtaactgacg acgctactgg tcagaaatat
1860ccgctggcag actatgcgct gactccggat atggcgattg tcgacgccaa
cctggttatg 1920gacatgccga agtccctgtg tgctttcggt ggtctggacg
cagtaactca cgccatggaa 1980gcttatgttt ctgtactggc atctgagttc
tctgatggtc aggctctgca ggcactgaaa 2040ctgctgaaag aatatctgcc
agcgtcctac cacgaagggt ctaaaaatcc ggtagcgcgt 2100gaacgtgttc
acagtgcagc gactatcgcg ggtatcgcgt ttgcgaacgc cttcctgggt
2160gtatgtcact caatggcgca caaactgggt tcccagttcc atattccgca
cggtctggca 2220aacgccctgc tgatttgtaa cgttattcgc tacaatgcga
acgacaaccc gaccaagcag 2280actgcattca gccagtatga ccgtccgcag
gctcgccgtc gttatgctga aattgccgac 2340cacttgggtc tgagcgcacc
gggcgaccgt actgctgcta agatcgagaa actgctggca 2400tggctggaaa
cgctgaaagc tgaactgggt attccgaaat ctatccgtga agctggcgtt
2460caggaagcag acttcctggc gaacgtggat aaactgtctg aagatgcatt
cgatgaccag 2520tgcaccggcg ctaacccgcg ttacccgctg atctccgagc
tgaaacagat tctgctggat 2580acctactacg gtcgtgatta tgtagaaggt
gaaactgcag cgaagaaaga agctgctccg 2640gctaaagctg agaaaaaagc
gaaaaaatcc gcttaa 267646891PRTEscherichia coli 46Met Ala Val Thr
Asn Val Ala Glu Leu Asn Ala Leu Val Glu Arg Val1 5 10 15Lys Lys Ala
Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln Val Asp 20 25 30Lys Ile
Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro 35 40 45Leu
Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val Glu Asp 50 55
60Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr65
70 75 80Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr Phe
Gly 85 90 95Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile
Val Pro 100 105 110Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser
Leu Ile Ser Leu 115 120 125Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro
His Pro Arg Ala Lys Asp 130 135 140Ala Thr Asn Lys Ala Ala Asp Ile
Val Leu Gln Ala Ala Ile Ala Ala145 150 155 160Gly Ala Pro Lys Asp
Leu Ile Gly Trp Ile Asp Gln Pro Ser Val Glu 165 170 175Leu Ser Asn
Ala Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala 180 185 190Thr
Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro 195 200
205Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr
210 215 220Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys
Thr Phe225 230 235 240Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser
Val Val Val Val Asp 245 250 255Ser Val Tyr Asp Ala Val Arg Glu Arg
Phe Ala Thr His Gly Gly Tyr 260 265 270Leu Leu Gln Gly Lys Glu Leu
Lys Ala Val Gln Asp Val Ile Leu Lys 275 280 285Asn Gly Ala Leu Asn
Ala Ala Ile Val Gly Gln Pro Ala Tyr Lys Ile 290 295 300Ala Glu Leu
Ala Gly Phe Ser Val Pro Glu Asn Thr Lys Ile Leu Ile305 310 315
320Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys
325 330
335Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala
340 345 350Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly
His Thr 355 360 365Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala
Arg Val Ser Tyr 370 375 380Phe Gly Gln Lys Met Lys Thr Ala Arg Ile
Leu Ile Asn Thr Pro Ala385 390 395 400Ser Gln Gly Gly Ile Gly Asp
Leu Tyr Asn Phe Lys Leu Ala Pro Ser 405 410 415Leu Thr Leu Gly Cys
Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn 420 425 430Val Gly Pro
Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala 435 440 445Glu
Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg 450 455
460Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly His
Lys465 470 475 480Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn
Asn Gly Tyr Ala 485 490 495Asp Gln Ile Thr Ser Val Leu Lys Ala Ala
Gly Val Glu Thr Glu Val 500 505 510Phe Phe Glu Val Glu Ala Asp Pro
Thr Leu Ser Ile Val Arg Lys Gly 515 520 525Ala Glu Leu Ala Asn Ser
Phe Lys Pro Asp Val Ile Ile Ala Leu Gly 530 535 540Gly Gly Ser Pro
Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu545 550 555 560His
Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile 565 570
575Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met
580 585 590Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr
Pro Phe 595 600 605Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr
Pro Leu Ala Asp 610 615 620Tyr Ala Leu Thr Pro Asp Met Ala Ile Val
Asp Ala Asn Leu Val Met625 630 635 640Asp Met Pro Lys Ser Leu Cys
Ala Phe Gly Gly Leu Asp Ala Val Thr 645 650 655His Ala Met Glu Ala
Tyr Val Ser Val Leu Ala Ser Glu Phe Ser Asp 660 665 670Gly Gln Ala
Leu Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu Pro Ala 675 680 685Ser
Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg Val His 690 695
700Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu
Gly705 710 715 720Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln
Phe His Ile Pro 725 730 735His Gly Leu Ala Asn Ala Leu Leu Ile Cys
Asn Val Ile Arg Tyr Asn 740 745 750Ala Asn Asp Asn Pro Thr Lys Gln
Thr Ala Phe Ser Gln Tyr Asp Arg 755 760 765Pro Gln Ala Arg Arg Arg
Tyr Ala Glu Ile Ala Asp His Leu Gly Leu 770 775 780Ser Ala Pro Gly
Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu Leu Ala785 790 795 800Trp
Leu Glu Thr Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg 805 810
815Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Asn Val Asp Lys Leu
820 825 830Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro
Arg Tyr 835 840 845Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp
Thr Tyr Tyr Gly 850 855 860Arg Asp Tyr Val Glu Gly Glu Thr Ala Ala
Lys Lys Glu Ala Ala Pro865 870 875 880Ala Lys Ala Glu Lys Lys Ala
Lys Lys Ser Ala 885 890472685DNAPantoea ananatis 47atggccgtta
ctaatgtcgc tgaactcaat gcactggttg aacgtgtaaa aaaagcccag 60caagaattcg
ccaatttttc tcaacaacag gtcgatgcca tcttccgcgc agccgcactg
120gccgccgcgg atgcccgaat tccactcgct aaaatggcgg tggcagaatc
gggcatgggc 180attgttgaag acaaagtcat taaaaatcac ttcgcttctg
aatacatcta caacgcctat 240aaggatgaga aaacctgcgg cgtactggac
accgatgata cgtttggcac catcacaatc 300gctgaaccca tcggcctgat
ttgcggtatc gtccccacca ctaaccctac ctccaccgcg 360attttcaagg
cacttatcag ccttaaaacc cgcaacggga ttatcttctc cccccatcct
420cgagccaaag atgcgacgaa caaagcggcg gatattgtcc tgcaggcagc
gattgccgct 480ggcgcgccca aagacattat aggctggatt gatgcacctt
ctgtggaact gtccaatcag 540ttgcaccatc ctgatattaa cctgattctg
gcgacgggtg gccccggcat ggtcaaagcc 600gcctacagct caggtaagcc
ggcgattggc gtgggggccg gtaacacgcc cgttgtcatc 660gatgaaacag
ctgatgttaa acgcgccgtt gcctccatcc tgatgtcaaa aacgtttgat
720aacggtgtga tctgtgcctc tgaacagtcg gttatcgtgg tggatgccgt
ctacgacgcc 780gtgcgcgagc gcttcgccag ccatggtggc tatttgcttc
agggacagga actgagtgcg 840gtacaaaata tcattctaaa aaacggtggg
cttaacgccg ccattgtggg ccagcctgcg 900gtgaagattg cggagatggc
cggcatcagc gtacctggtg aaaccaaaat cctgattggc 960gaagttgaac
gggtcgatga atcagagcct ttcgctcatg aaaaactgtc gccgacactg
1020gcgatgtacc gtgctaaaga ttatcaggat gccgtcagca aagcggagaa
actggtggcg 1080atgggtggta ttggtcatac gtcatgcctg tataccgacc
aggacaatca gacagcgcgc 1140gtgcactatt ttggcgacaa gatgaaaaca
gcccgcattc tgatcaacac gccagcttct 1200cagggcggta ttggtgattt
atataacttc aaactcgccc cttctctgac actgggttgt 1260ggttcctggg
gcggtaactc catttctgaa aacgtggggc ccaaacatct catcaacaag
1320aaaaccgtcg ctaagcgagc tgaaaatatg ttgtggcata aacttccgaa
gtccatttac 1380ttccgtcgcg gctctttacc cattgcgctt gaagagatcg
ccaccgacgg tgccaaacgc 1440gcgtttgtgg tgactgaccg cttcctgttt
aacaacggtt atgccgatca ggtcacccgc 1500gttttaaaat ctcacggcat
cgaaaccgaa gttttctttg aggttgaagc ggatcccacc 1560ttaagcatcg
tgcgtaaagg tgcagaacag atgaacagct ttaagccaga cgtgatcatc
1620gccctgggcg gcggttcgcc gatggatgca gccaaaatca tgtgggtcat
gtatgagcat 1680cctgaaaccc attttgaaga gctggcactg cggtttatgg
atattcgcaa acgtatctat 1740aagttcccta aaatgggcgt gaaagcgcgc
atggtggcca ttacgacaac ctcaggcaca 1800ggttcagaag tgacgccttt
tgccgtggta acggatgacg cgaccggaca gaaatacccg 1860ctggccgatt
atgcgctgac gccggatatg gctatcgttg atgccaacct ggtcatggat
1920atgccacgtt cactctgtgc cttcggcggt ctggatgcgg tgacgcacgc
gctggaagcc 1980tatgtgtccg tcctggccaa tgaatactcc gatggtcagg
ccctgcaggc gcttaagctg 2040cttaaagaga acttaccggc gagttatgca
gaaggtgcaa aaaatccggt tgcccgtgaa 2100cgtgtacata atgccgccac
catcgccggt atcgcctttg cgaacgcctt cctcggggtt 2160tgtcactcaa
tggcgcataa gcttggctct gagttccata ttcctcatgg actggctaac
2220tcgctgctga tttccaacgt tattcgctat aacgccaatg acaaccctac
taagcaaacc 2280gcattcagcc agtacgatcg tccccaggcg cgtcgtcgtt
atgctgaaat tgcggatcat 2340cttggtctca ccgcgccggg cgaccgcact
gcccagaaaa ttgagaagct gctggtatgg 2400ctggatgaaa tcaaaacgga
actgggtatt ccggcctcaa ttcgtgaagc cggtgtgcag 2460gaggctgact
tcctggcgaa agtcgataaa ctggcggatg atgcctttga tgaccagtgt
2520actggcgcga atccacgtta tccgctgatt gccgaactca aacagctgat
gctggacagc 2580tactacggac gcaaatttgt cgagccgttc gccagtgccg
ccgaggctgc ccaggctcag 2640cctgtcagtg acagcaaagc ggcgaagaaa
gctaaaaaag cctaa 268548894PRTPantoea ananatis 48Met Ala Val Thr Asn
Val Ala Glu Leu Asn Ala Leu Val Glu Arg Val1 5 10 15Lys Lys Ala Gln
Gln Glu Phe Ala Asn Phe Ser Gln Gln Gln Val Asp 20 25 30Ala Ile Phe
Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro 35 40 45Leu Ala
Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val Glu Asp 50 55 60Lys
Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr65 70 75
80Lys Asp Glu Lys Thr Cys Gly Val Leu Asp Thr Asp Asp Thr Phe Gly
85 90 95Thr Ile Thr Ile Ala Glu Pro Ile Gly Leu Ile Cys Gly Ile Val
Pro 100 105 110Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ala Leu
Ile Ser Leu 115 120 125Lys Thr Arg Asn Gly Ile Ile Phe Ser Pro His
Pro Arg Ala Lys Asp 130 135 140Ala Thr Asn Lys Ala Ala Asp Ile Val
Leu Gln Ala Ala Ile Ala Ala145 150 155 160Gly Ala Pro Lys Asp Ile
Ile Gly Trp Ile Asp Ala Pro Ser Val Glu 165 170 175Leu Ser Asn Gln
Leu His His Pro Asp Ile Asn Leu Ile Leu Ala Thr 180 185 190Gly Gly
Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala 195 200
205Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr Ala
210 215 220Asp Val Lys Arg Ala Val Ala Ser Ile Leu Met Ser Lys Thr
Phe Asp225 230 235 240Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val
Ile Val Val Asp Ala 245 250 255Val Tyr Asp Ala Val Arg Glu Arg Phe
Ala Ser His Gly Gly Tyr Leu 260 265 270Leu Gln Gly Gln Glu Leu Ser
Ala Val Gln Asn Ile Ile Leu Lys Asn 275 280 285Gly Gly Leu Asn Ala
Ala Ile Val Gly Gln Pro Ala Val Lys Ile Ala 290 295 300Glu Met Ala
Gly Ile Ser Val Pro Gly Glu Thr Lys Ile Leu Ile Gly305 310 315
320Glu Val Glu Arg Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys Leu
325 330 335Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Tyr Gln Asp
Ala Val 340 345 350Ser Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile
Gly His Thr Ser 355 360 365Cys Leu Tyr Thr Asp Gln Asp Asn Gln Thr
Ala Arg Val His Tyr Phe 370 375 380Gly Asp Lys Met Lys Thr Ala Arg
Ile Leu Ile Asn Thr Pro Ala Ser385 390 395 400Gln Gly Gly Ile Gly
Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser Leu 405 410 415Thr Leu Gly
Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn Val 420 425 430Gly
Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala Glu 435 440
445Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg Gly
450 455 460Ser Leu Pro Ile Ala Leu Glu Glu Ile Ala Thr Asp Gly Ala
Lys Arg465 470 475 480Ala Phe Val Val Thr Asp Arg Phe Leu Phe Asn
Asn Gly Tyr Ala Asp 485 490 495Gln Val Thr Arg Val Leu Lys Ser His
Gly Ile Glu Thr Glu Val Phe 500 505 510Phe Glu Val Glu Ala Asp Pro
Thr Leu Ser Ile Val Arg Lys Gly Ala 515 520 525Glu Gln Met Asn Ser
Phe Lys Pro Asp Val Ile Ile Ala Leu Gly Gly 530 535 540Gly Ser Pro
Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu His545 550 555
560Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile Arg
565 570 575Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Arg
Met Val 580 585 590Ala Ile Thr Thr Thr Ser Gly Thr Gly Ser Glu Val
Thr Pro Phe Ala 595 600 605Val Val Thr Asp Asp Ala Thr Gly Gln Lys
Tyr Pro Leu Ala Asp Tyr 610 615 620Ala Leu Thr Pro Asp Met Ala Ile
Val Asp Ala Asn Leu Val Met Asp625 630 635 640Met Pro Arg Ser Leu
Cys Ala Phe Gly Gly Leu Asp Ala Val Thr His 645 650 655Ala Leu Glu
Ala Tyr Val Ser Val Leu Ala Asn Glu Tyr Ser Asp Gly 660 665 670Gln
Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Asn Leu Pro Ala Ser 675 680
685Tyr Ala Glu Gly Ala Lys Asn Pro Val Ala Arg Glu Arg Val His Asn
690 695 700Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu
Gly Val705 710 715 720Cys His Ser Met Ala His Lys Leu Gly Ser Glu
Phe His Ile Pro His 725 730 735Gly Leu Ala Asn Ser Leu Leu Ile Ser
Asn Val Ile Arg Tyr Asn Ala 740 745 750Asn Asp Asn Pro Thr Lys Gln
Thr Ala Phe Ser Gln Tyr Asp Arg Pro 755 760 765Gln Ala Arg Arg Arg
Tyr Ala Glu Ile Ala Asp His Leu Gly Leu Thr 770 775 780Ala Pro Gly
Asp Arg Thr Ala Gln Lys Ile Glu Lys Leu Leu Val Trp785 790 795
800Leu Asp Glu Ile Lys Thr Glu Leu Gly Ile Pro Ala Ser Ile Arg Glu
805 810 815Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Lys Val Asp Lys
Leu Ala 820 825 830Asp Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn
Pro Arg Tyr Pro 835 840 845Leu Ile Ala Glu Leu Lys Gln Leu Met Leu
Asp Ser Tyr Tyr Gly Arg 850 855 860Lys Phe Val Glu Pro Phe Ala Ser
Ala Ala Glu Ala Ala Gln Ala Gln865 870 875 880Pro Val Ser Asp Ser
Lys Ala Ala Lys Lys Ala Lys Lys Ala 885 890492676DNAPectobacterium
atrosepticum 49atggccgtaa ccaacgttgc tgaacttaac gcactggtcg
aaagagttaa aaaggcacag 60caggaatttg ccacttacac tcaggaacaa gtggacaaga
tcttccgcgc tgccgcactc 120gctgcatcgg atgcccgtat cccgctggca
aaaatggcgg ttgctgaatc cggtatgggg 180atcgtggaag ataaagtcat
caaaaaccac ttcgcatccg aatacattta taacgcctat 240caggatgaaa
aaacctgtgg cgtcctctct actgatgaca ctttcggtac tattaccatt
300gcagagccta ttggcctgat ttgcggtatt gttcccacca ccaaccccac
ttctaccgcg 360atttttaaag cgctgatcag cctgaagact cgtaacggga
ttatcttctc tccccatcca 420cgtgcaaaaa atgcgaccaa taaagccgca
gacattgtac tgcaagctgc gattgccgct 480ggcgccccga aagatatcat
cggctggatt gatcaaccgt ccgtcgattt atccaaccaa 540ctgatgcacc
acccagatat caacctgatt ctggctaccg gcgggccggg tatggtgaaa
600gcggcataca gctcaggtaa accggcgatc ggcgtaggtg caggtaacac
ccccgttgtt 660attgacgaaa cggcggatat taagcgtgcc gttgcctcta
tcctgatgtc gaaaaccttc 720gataacggcg tcatttgtgc gtcagaacag
tcagtcatcg tggtagacag cgcctatgat 780gccgtacgtg agcgtttcgc
cacccacggc ggctacatgc tgaaaggcaa agaacttcat 840gccgtacaag
gcattctgct gaaaaacggc tcactgaatg ccgacattgt gggccagcca
900gcaccaaaga tcgctgaaat ggcgggtatc accgtccctg cgaacaccaa
agtgctgatc 960ggtgaagtga cggccgttga tgaatccgaa ccgtttgccc
atgaaaaact gtctccgacg 1020ctggcgatgt accgggcgaa agacttcaat
gacgccgtca ttaaagcgga aaaactggtg 1080gcaatgggtg gcatcggtca
cacatcctgc ctgtataccg atcaggacaa tcagccagag 1140cgtgtaaatc
atttcgggaa tatgatgaaa acggcacgta tcctgattaa cacgccggct
1200tctcagggtg gtatcggcga tctctacaac ttcaaactcg ctccgtctct
gacactgggc 1260tgtggctcat ggggcggaaa ctccatctcc gaaaacgtcg
gtccgaagca cttgatcaac 1320aaaaaaacgg tagccaagcg agcagagaat
atgttgtggc ataaacttcc taaatccatt 1380tacttccgtc gtggctcact
gcctatcgca cttgaagaag tcgcatccga tggtgcaaaa 1440cgcgcattta
tcgtgactga ccgcttcctg ttcaataatg gctacgttga tcaggtaact
1500tccgtactga aacaacacgg actggaaacc gaagttttct ttgaagttga
agctgacccg 1560acactgagca tcgtgcgcaa aggcgcggaa caaatgcact
ccttcaagcc cgatgtgatt 1620attgcactgg gtggcggttc tccgatggat
gctgcgaaga tcatgtgggt gatgtatgag 1680caccctacta cacacttcga
agagctggcg ctgcgcttta tggatatccg taaacgtatc 1740tataagttcc
cgaaaatggg tgtcaaagcc aagatggtgg cgattaccac tacatccggt
1800actggttccg aagtcacgcc atttgccgtg gtgaccgacg atgcaactgg
acagaaatat 1860ccgttggcgg actatgcgct gaccccagat atggccattg
ttgatgccaa tctggtgatg 1920aacatgccga aatcgctgtg tgcctttggt
gggctcgatg ccgtaaccca ctcgctggaa 1980gcctatgttt ccgtgctggc
aaatgaatat tcagacggac aggcgttaca agcgctgaaa 2040ctgctgaagg
aaaatctgcc ggacagctac cgtgacggtg cgaaaaaccc ggttgcccgt
2100gagcgcgtgc acaacgccgc gacgattgcg ggtatcgcgt ttgccaacgc
cttcctcggc 2160gtctgtcact caatggcgca taaactgggc tcggagttcc
atattccgca cggtctggct 2220aatgccatgc tgatctcgaa cgtgattcgc
tataacgcga acgataaccc gaccaaacaa 2280acaacgttca gccaatatga
ccgtccgcaa gctcgtcgtc gttacgctga aatagccgac 2340cacctacgtt
tgactgcgcc tagcgaccgt actgcacaga aaatcgagaa attactgaac
2400tggctggaag aaataaagac cgaactgggg atcccagcgt ccattcgtga
agcgggcgta 2460caggaggccg atttcctggc taaggtcgat aaactgtcag
aagatgcgtt cgacgatcag 2520tgtactggtg ctaacccacg ctacccgctg
atttctgaat tgaaacagat tctgctggac 2580acttactatg gtcgtaagtt
ctctgaagag gtaaaaacgg aaaccgttga acctgtagca 2640aaagccgcca
aaaccggcaa gaaagccgca cattaa 267650891PRTPectobacterium
atrosepticum 50Met Ala Val Thr Asn Val Ala Glu Leu Asn Ala Leu Val
Glu Arg Val1 5 10 15Lys Lys Ala Gln Gln Glu Phe Ala Thr Tyr Thr Gln
Glu Gln Val Asp 20 25 30Lys Ile Phe Arg Ala Ala Ala Leu Ala Ala Ser
Asp Ala Arg Ile Pro 35 40 45Leu Ala Lys Met Ala Val Ala Glu Ser Gly
Met Gly Ile Val Glu Asp 50 55 60Lys Val Ile Lys Asn His Phe Ala Ser
Glu Tyr Ile Tyr Asn Ala Tyr65 70 75 80Gln Asp Glu Lys Thr Cys Gly
Val Leu Ser Thr Asp Asp Thr Phe Gly 85 90 95Thr Ile Thr Ile Ala Glu
Pro Ile Gly Leu Ile Cys Gly Ile Val Pro 100 105 110Thr Thr Asn Pro
Thr Ser Thr Ala Ile
Phe Lys Ala Leu Ile Ser Leu 115 120 125Lys Thr Arg Asn Gly Ile Ile
Phe Ser Pro His Pro Arg Ala Lys Asn 130 135 140Ala Thr Asn Lys Ala
Ala Asp Ile Val Leu Gln Ala Ala Ile Ala Ala145 150 155 160Gly Ala
Pro Lys Asp Ile Ile Gly Trp Ile Asp Gln Pro Ser Val Asp 165 170
175Leu Ser Asn Gln Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala
180 185 190Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly
Lys Pro 195 200 205Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val
Ile Asp Glu Thr 210 215 220Ala Asp Ile Lys Arg Ala Val Ala Ser Ile
Leu Met Ser Lys Thr Phe225 230 235 240Asp Asn Gly Val Ile Cys Ala
Ser Glu Gln Ser Val Ile Val Val Asp 245 250 255Ser Ala Tyr Asp Ala
Val Arg Glu Arg Phe Ala Thr His Gly Gly Tyr 260 265 270Met Leu Lys
Gly Lys Glu Leu His Ala Val Gln Gly Ile Leu Leu Lys 275 280 285Asn
Gly Ser Leu Asn Ala Asp Ile Val Gly Gln Pro Ala Pro Lys Ile 290 295
300Ala Glu Met Ala Gly Ile Thr Val Pro Ala Asn Thr Lys Val Leu
Ile305 310 315 320Gly Glu Val Thr Ala Val Asp Glu Ser Glu Pro Phe
Ala His Glu Lys 325 330 335Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala
Lys Asp Phe Asn Asp Ala 340 345 350Val Ile Lys Ala Glu Lys Leu Val
Ala Met Gly Gly Ile Gly His Thr 355 360 365Ser Cys Leu Tyr Thr Asp
Gln Asp Asn Gln Pro Glu Arg Val Asn His 370 375 380Phe Gly Asn Met
Met Lys Thr Ala Arg Ile Leu Ile Asn Thr Pro Ala385 390 395 400Ser
Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser 405 410
415Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn
420 425 430Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys
Arg Ala 435 440 445Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile
Tyr Phe Arg Arg 450 455 460Gly Ser Leu Pro Ile Ala Leu Glu Glu Val
Ala Ser Asp Gly Ala Lys465 470 475 480Arg Ala Phe Ile Val Thr Asp
Arg Phe Leu Phe Asn Asn Gly Tyr Val 485 490 495Asp Gln Val Thr Ser
Val Leu Lys Gln His Gly Leu Glu Thr Glu Val 500 505 510Phe Phe Glu
Val Glu Ala Asp Pro Thr Leu Ser Ile Val Arg Lys Gly 515 520 525Ala
Glu Gln Met His Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly 530 535
540Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr
Glu545 550 555 560His Pro Thr Thr His Phe Glu Glu Leu Ala Leu Arg
Phe Met Asp Ile 565 570 575Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met
Gly Val Lys Ala Lys Met 580 585 590Val Ala Ile Thr Thr Thr Ser Gly
Thr Gly Ser Glu Val Thr Pro Phe 595 600 605Ala Val Val Thr Asp Asp
Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp 610 615 620Tyr Ala Leu Thr
Pro Asp Met Ala Ile Val Asp Ala Asn Leu Val Met625 630 635 640Asn
Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr 645 650
655His Ser Leu Glu Ala Tyr Val Ser Val Leu Ala Asn Glu Tyr Ser Asp
660 665 670Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Asn Leu
Pro Asp 675 680 685Ser Tyr Arg Asp Gly Ala Lys Asn Pro Val Ala Arg
Glu Arg Val His 690 695 700Asn Ala Ala Thr Ile Ala Gly Ile Ala Phe
Ala Asn Ala Phe Leu Gly705 710 715 720Val Cys His Ser Met Ala His
Lys Leu Gly Ser Glu Phe His Ile Pro 725 730 735His Gly Leu Ala Asn
Ala Met Leu Ile Ser Asn Val Ile Arg Tyr Asn 740 745 750Ala Asn Asp
Asn Pro Thr Lys Gln Thr Thr Phe Ser Gln Tyr Asp Arg 755 760 765Pro
Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Arg Leu 770 775
780Thr Ala Pro Ser Asp Arg Thr Ala Gln Lys Ile Glu Lys Leu Leu
Asn785 790 795 800Trp Leu Glu Glu Ile Lys Thr Glu Leu Gly Ile Pro
Ala Ser Ile Arg 805 810 815Glu Ala Gly Val Gln Glu Ala Asp Phe Leu
Ala Lys Val Asp Lys Leu 820 825 830Ser Glu Asp Ala Phe Asp Asp Gln
Cys Thr Gly Ala Asn Pro Arg Tyr 835 840 845Pro Leu Ile Ser Glu Leu
Lys Gln Ile Leu Leu Asp Thr Tyr Tyr Gly 850 855 860Arg Lys Phe Ser
Glu Glu Val Lys Thr Glu Thr Val Glu Pro Val Ala865 870 875 880Lys
Ala Ala Lys Thr Gly Lys Lys Ala Ala His 885 890512679DNASalmonella
enterica 51atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgtgtaaa
aaaagcccag 60cgtgaatatg ccagtttcac tcaagaacag gtcgacaaaa tcttccgcgc
cgccgctctg 120gcagccgctg atgcccgcat tccgctggcc aaaatggccg
tcgccgaatc aggtatgggt 180atcgtggaag acaaagtgat taaaaaccac
ttcgcttctg aatatattta caatgcctat 240aaagatgaaa aaacctgcgg
cgtgctgtca gaagacgaca ccttcgggac catcaccatt 300gctgaaccta
tcggcattat ttgcggtatc gttccaacca ctaacccgac ctctactgcg
360atcttcaaat cgctgattag cctgaagacc cgtaacgcca tcatcttttc
tccgcatccg 420cgcgctaaag aagcaactaa caaagcggca gacatcgttc
tgcaagcggc tatcgctgcc 480ggcgcaccga aagatctgat tggctggatc
gatcaacctt ccgtagaact gtctaatgcg 540ctgatgcacc acccggatat
taacctgatc ctcgccactg gcggtccagg catggttaaa 600gctgcataca
gctccggtaa accggcaatc ggcgtaggcg caggtaacac cccggttgtc
660attgatgaaa ccgctgatat caaacgcgct gtggcgtctg ttctgatgtc
taaaaccttc 720gataacggcg taatctgtgc ttctgaacag tctgttgtcg
ttgttgattc cgtctatgat 780gccgttcgcg aacgtttcgc cagccacggc
ggctacatgc tgcagggcca ggagctgaaa 840gcggttcaaa acgttattct
gaaaaatggc gctctgaacg ccgctatcgt cggtcagcca 900gcctacaaaa
tcgctgaact ggcaggcttc tccgtaccag aaaccaccaa gattctgatc
960ggtgaagtta cggtcgttga cgaaagcgaa ccgttcgcac acgaaaaact
gtctccgact 1020ctagcgatgt accgtgcgaa agatttcgaa gaagcggtag
aaaaagcaga aaaactggtc 1080gctatgggcg gtatcggtca cacctcctgc
ctgtacactg accaggataa ccagccagaa 1140cgcgttgctt acttcggtca
gatgatgaaa accgcgcgta tcctgatcaa caccccggcc 1200tctcagggtg
gtatcggtga cctgtacaac ttcaaactcg caccttccct gacgttgggt
1260tgtggttcct ggggtggtaa ctccatctct gaaaacgttg gtccgaaaca
cctgatcaac 1320aagaaaaccg ttgctaagcg agctgaaaac atgttgtggc
acaaacttcc gaaatctatc 1380tacttccgcc gtggctctct gcccatcgcg
ctggatgaag tgattactga tggccacaaa 1440cgtgcgctca tcgtgactga
ccgcttcctg ttcaacaacg gctatgcaga ccagatcacc 1500tctgtgctga
aagcggctgg cgttgaaacc gaagtcttct tcgaagttga agcagacccg
1560acgctttccg ttgttcgcaa aggcgctgag ctggctaact ccttcaaacc
ggacgtgatc 1620atcgcgctgg gcggcggttc cccgatggac gccgcgaaaa
tcatgtgggt catgtacgaa 1680catccggaaa ctcacttcga agaactggcg
ctgcgcttta tggacatccg taaacgtatc 1740tacaagttcc cgaaaatggg
cgtgaaagcg aaaatgatcg ccgtcaccac cacttccggt 1800accggttctg
aagtcacacc gtttgcggtt gtgaccgaca atgcaaccgg tcagaaatat
1860ccgctggctg actatgccct gaccccggat atggcgattg tcgatgccaa
cctggtgatg 1920gatatgccga agtccctgtg tgcgttcggt ggtctggatg
cggtaactca cgccctggaa 1980gcttacgttt ccgtactggc ttctgagttc
tctgacggtc aggctctgca ggctctgaaa 2040ctgctgaaag aaaacctgcc
ggcgtcttac cacgaagggt ctaaaaaccc ggttgcgcgt 2100gaacgtgttc
acagtgcagc gactatcgcc ggtatcgcgt ttgccaacgc cttcctcggt
2160gtatgtcact ccatggcgca caaactgggc tctcagttcc acattccgca
cggtctggcg 2220aacgccctgc tgatttgtaa cgttatccgc tacaacgcga
atgacaaccc gaccaagcag 2280accgctttca gccagtacga tcgtccgcag
gcacgccgtc gttacgctga aattgctgac 2340cacctgggcc tgagcgcgcc
gggcgaccgt accgccgcta agattgaaaa actgctggca 2400tggctggaaa
gcattaaagc tgagctgggc attcctaagt ctatacgtga agcaggcgtg
2460caggaagctg acttcctggc acacgttgac aaactgtctg aagatgcctt
cgatgaccag 2520tgcaccggcg ctaacccgcg ttatccgctg atctccgaac
tgaaacagat tctgctggat 2580acctactacg gtcgtgattt caccgaaggt
gaagttgcag cgaagaaaga cgtcgttgcc 2640gcaccgaaag cagagaaaaa
agcgaaaaaa tccgcttaa 267952892PRTSalmonella enterica 52Met Ala Val
Thr Asn Val Ala Glu Leu Asn Ala Leu Val Glu Arg Val1 5 10 15Lys Lys
Ala Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln Val Asp 20 25 30Lys
Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro 35 40
45Leu Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val Glu Asp
50 55 60Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala
Tyr65 70 75 80Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp
Thr Phe Gly 85 90 95Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys
Gly Ile Val Pro 100 105 110Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe
Lys Ser Leu Ile Ser Leu 115 120 125Lys Thr Arg Asn Ala Ile Ile Phe
Ser Pro His Pro Arg Ala Lys Glu 130 135 140Ala Thr Asn Lys Ala Ala
Asp Ile Val Leu Gln Ala Ala Ile Ala Ala145 150 155 160Gly Ala Pro
Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser Val Glu 165 170 175Leu
Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala 180 185
190Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro
195 200 205Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp
Glu Thr 210 215 220Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met
Ser Lys Thr Phe225 230 235 240Asp Asn Gly Val Ile Cys Ala Ser Glu
Gln Ser Val Val Val Val Asp 245 250 255Ser Val Tyr Asp Ala Val Arg
Glu Arg Phe Ala Ser His Gly Gly Tyr 260 265 270Met Leu Gln Gly Gln
Glu Leu Lys Ala Val Gln Asn Val Ile Leu Lys 275 280 285Asn Gly Ala
Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Tyr Lys Ile 290 295 300Ala
Glu Leu Ala Gly Phe Ser Val Pro Glu Thr Thr Lys Ile Leu Ile305 310
315 320Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala His Glu
Lys 325 330 335Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe
Glu Glu Ala 340 345 350Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly
Gly Ile Gly His Thr 355 360 365Ser Cys Leu Tyr Thr Asp Gln Asp Asn
Gln Pro Glu Arg Val Ala Tyr 370 375 380Phe Gly Gln Met Met Lys Thr
Ala Arg Ile Leu Ile Asn Thr Pro Ala385 390 395 400Ser Gln Gly Gly
Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser 405 410 415Leu Thr
Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn 420 425
430Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala
435 440 445Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe
Arg Arg 450 455 460Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr
Asp Gly His Lys465 470 475 480Arg Ala Leu Ile Val Thr Asp Arg Phe
Leu Phe Asn Asn Gly Tyr Ala 485 490 495Asp Gln Ile Thr Ser Val Leu
Lys Ala Ala Gly Val Glu Thr Glu Val 500 505 510Phe Phe Glu Val Glu
Ala Asp Pro Thr Leu Ser Val Val Arg Lys Gly 515 520 525Ala Glu Leu
Ala Asn Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly 530 535 540Gly
Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu545 550
555 560His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp
Ile 565 570 575Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys
Ala Lys Met 580 585 590Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser
Glu Val Thr Pro Phe 595 600 605Ala Val Val Thr Asp Asn Ala Thr Gly
Gln Lys Tyr Pro Leu Ala Asp 610 615 620Tyr Ala Leu Thr Pro Asp Met
Ala Ile Val Asp Ala Asn Leu Val Met625 630 635 640Asp Met Pro Lys
Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr 645 650 655His Ala
Leu Glu Ala Tyr Val Ser Val Leu Ala Ser Glu Phe Ser Asp 660 665
670Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Asn Leu Pro Ala
675 680 685Ser Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg
Val His 690 695 700Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn
Ala Phe Leu Gly705 710 715 720Val Cys His Ser Met Ala His Lys Leu
Gly Ser Gln Phe His Ile Pro 725 730 735His Gly Leu Ala Asn Ala Leu
Leu Ile Cys Asn Val Ile Arg Tyr Asn 740 745 750Ala Asn Asp Asn Pro
Thr Lys Gln Thr Ala Phe Ser Gln Tyr Asp Arg 755 760 765Pro Gln Ala
Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Gly Leu 770 775 780Ser
Ala Pro Gly Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu Leu Ala785 790
795 800Trp Leu Glu Ser Ile Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile
Arg 805 810 815Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala His Val
Asp Lys Leu 820 825 830Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly
Ala Asn Pro Arg Tyr 835 840 845Pro Leu Ile Ser Glu Leu Lys Gln Ile
Leu Leu Asp Thr Tyr Tyr Gly 850 855 860Arg Asp Phe Thr Glu Gly Glu
Val Ala Ala Lys Lys Asp Val Val Ala865 870 875 880Ala Pro Lys Ala
Glu Lys Lys Ala Lys Lys Ser Ala 885 890
* * * * *